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RUBRIK VERSAL I Unterrubrik

02 I 2015 CHF 24,90 I E 23,80 www.chemieplus.ch

START-UP: DRUGS FROM HAIRY ROOTS  P. 26

TISSUE ENGINEERING: PERIODONTITIS – THE INSIDIOUS DISEASE   P. 17

NOVEL FUNCTIONAL BIOASSAYS TO ACCELERATE BIOLOGICS DEVELOPMENT  P. 60

INNOVATIONS IN MEDTECH: PERSONALIZED MEDICAL TECHNOLOGY  P. 10

Cancer Research:

Can Drug Resistance be Overcome?   P. 6 Life Sciences plus 01 I 2015

1


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EDITORIAL

“The important thing is to not stop questioning.” Albert Einstein

Innovation always sells

P

ersonalized Medicine is a famous buzzword from

There are many innovative minds in Switzerland, having

pharmaceutical industry and usually means individ-

innovative ideas (see page 22 ff.). Some of these concepts

ual drugs. Actually, personalized medicine is more than

make it to the market, others have to be abandoned. Some-

drug research: Fundamental research in biology, physiolo-

times, economy decides about success and failure of a

gy and medicine is necessary to understand the molecular

certain venture, since there is no project without funding.

mechanisms of a disease like cancer. The tons of findings

Interestingly, the un-willingness to invest and tense finan-

need to be screened and analysed which requires help of

cial situations in some cases stimulate innovative capabi-

computational science. Thus, personalized medicine is not

lity even more. In a mature market for example, only pio-

pharmaceutical research but an extensive, interdiscipli-

neering ideas attract attention. Thus, ground breaking

nary science which requires experts in all disciplines. The

findings always sell.

next step after understanding mechanisms is invention. For innovative ideas have to be found in order to push ap-

But there is more to come – more to discover, to decipher

plied research and technology further ahead.

and to understand. The interactions of humans with their environment or the biochemical processes within a single

Regarding medicine there is much room for innovative

person on molecular or even smaller level are still full of

technology. This includes technology for the application

mysteries. A famous quote of Albert Einstein says: “The

bedside, directly on the patient, as well as technical equip-

important thing is to not stop questioning. Curiosity has its

ment for diagnostics or research: Sensors are needed to

own reason for existence. One cannot help but be in awe

monitor physiological conditions, prostheses become more

when he contemplates the mysteries of eternity, of life, of

and more intelligent or individual (see page 10), cancer

the marvellous structure of reality. It is enough if one tries

treatments become more effective (see page 6) or defective

merely to comprehend a little of this mystery each day.”

tissue can be regenerated (see page 17). The development of such powerful gadgets improving medical treatments is

With this in mind, the articles of this issue of “lifesciences

not only life science but science for lives. This science aims

plus” gain even more importance: Not only the successful

to safe lives or make life for people with ill-health more

results of a research project are applaudable, but also the

worth living.

commitment of researchers and developers to their daily work. No pain, no gain! Projects only succeed with people willing to give their time and effort. Luckily, there are a lot of scientists feeling the same way about their work as Einstein described. They are in awe and they are curious. And therefore, they don’t stop doing research. Hence, Switzer-

ur opinion o y , s r e d a e r Dear to us. is important hemieplus.ch/lifesciencesplus w e link www.c about the ne Please use th d suggestions an s ew vi re ur to pass on yo magazine.

land should be proud of its research and development institutions, which build important pillars of economy and reputation worldwide.

SONJA BICHSEL-KÄSER Editor


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S U M M A RY

MEDTECH

10

Which developments,

possibilities and limitations have an impact on the medtech industry in Switzerland? Personalized medicine is not only a buzzword from the pharmaceutical industry but also from medtech industry. Researchers and industrial partners develop various devices and gadgets to monitor bodily functions. But many projects lack investors.

6 CANCER RESEARCH

Malignant tumours

may be resistant towards certain chemotherapeutics. In cancer research, personalized medicine is sometimes the only chance to find a cure. In order to overcome a resistance, genetic basics have to be known to understand the whole “tumour ecosystem”. Only then the most effective chemotherapeutics can be found.

SWISS RESEARCH

04 News 17

  Tissue Engineering: Periodontitis – The Insidious Disease

22  Drug Research & Development: Fighting Tuberculosis by Restoring Ethionamide’s Antibiotic Power Selective Inhibitors

26

 Phytopharmaceuticals: Drugs from Hairy Roots

PLAYERS & PRODUCTS

38

  Advanced Monitoring and Display of Cell-Based Assays in a Microplate Reader

40

  High Throughput Screening: Research for Oncological Pharmaceutical Compounds with 3-D Spheroid Cultures

42

  Bioprocessing: Tailored Cell Culture Media – Produced in a Cleanroom of GMP Standard

46

  Cell Culture: Paving the Way for Cell Fate Discovery LAB & PROCESS

14

SLEEP RESEARCH

Brain activity and sleep phases can be visualised by EEG. By applying the method, researchers from the University of Zurich found out that genetic variation influences sleep characteristics. EEG patterns also change with brain development stages in children. A map of the active brain regions can give information about the stage of development.

30

  Bioimaging: Illuminating the Future Using CytoViva Hyperspectral Microscopy

34  Metabolomics: Measuring Glycolytic Function in Immune Cells

48

  Uncover and Handle Details at the Nanometer Scale

49  Improved Cryo Sample Stability 50 Tools 52  Tools at MipTec

36

  Food Analysis: Serial Dilutions in Cost Effective Single-Use Sterile Bags

FILTRATE

62 News 65  Edition Notice

Lifesciences plus 02 I 2015

3


SWISS RESEARCH

NEWS

Escherichia coli cells (Picture: Wikimedia)

Flu remedies help combat E. coli bacteria

Trillions of bacteria

Ebola Virus Particle (Picture: iStock)

Ebola vaccine tested successfully for the first time

The Ebola virus

disease epidemic in West Africa has not been defeated yet, although the number of cases has dropped substantially since the start of the year. Just one Ebola case in the most heavily affected countries, Guinea, Liberia and Sierra Leone, can lead to a resurgence. Two vaccines that were recently developed have already undergone preliminary tests in humans. One of the vaccines, “rVSV-Zebov”, has now been tested in the first large field trial of efficacy and effectiveness in Guinea, West Africa. Prof. Dr. Matthias Egger from the Institute of Social and Preventive Medicine at the University of Bern was involved in this “Ebola ça suffit” trial, together with PD Dr. Sven Trelle and other staff from the Clinical Trials Unit CTU Bern at the University’s clinical study centre and Bern University Hospital. The initial results of the study show that the vaccine can effectively contain the further spread of the Ebola virus. The results of the field study and the innovative study methods were published in the “Lancet” and “BMJ” scientific journals. The international group of researchers used a multi-stage approach based on “ring vaccination”, which was used to eradicate smallpox in Africa in the 1970s, to test the efficacy of the vaccine. “We can say that the vaccine offers 100 % protection against Ebola after roughly one week,” Sven Trelle says. Overall the protective effect within the rings, in which there were also people who had not been vaccinated, was still 76 percent, which means the transmission of the virus could be interrupted in many cases. “It is not just the efficacy of the Ebola vaccine that has now been shown but also the effectiveness of the ring vaccination strategy,” Egger explains with delight. “This could finally be the beginning of the end of the Ebola epidemic in West Africa and also be useful when combating this disease in the future.” #  www.unibe.ch

populate the human gut – which makes them more common than any other cells in our body. The composition of this bacterial population is very variable and influenced by our diet. If entire bacterial groups suddenly multiply heavily, critical situations occur. They damage the intestinal tissue and cause inflammations. How such shifts are triggered largely remained a mystery. Physiologists from the University of Zurich have now discovered why the intestinal bacterium Escherichia coli (E. coli) multiplies heavily and has an inflammatory effect. In their normal state, E. coli are harmless and only make up around 0.1 % of the intestinal flora. In large amounts, however, they can cause diarrhea or a serious intestinal inflammation. The Zurich study reveals that an overproduction of E. coli can be attributed to the availability of the carbohydrate sialic acid. In order to utilize the sialic acid, the enzyme sialidase is needed, which is released by other intestinal bacteria, but, strikingly, not by E. coli itself. Professor Thierry Hennet from the University of Zurich and his colleagues succeeded in demonstrating that an injury to the intestinal mucosa initially causes the increased multiplication of a non-pathogenic bacteria, which emits sialidase. This increased enzyme production releases sialic acid, which facilitates an overproduction of E. coli and can thus cause intestinal inflammation. The researchers also discovered that the intake of a sialidase inhibitor prevents the excessive formation of E. coli and was thus able to alleviate the disease symptoms. Interestingly, such sialidase inhibitors were already developed against the influenza virus. “Derivatives of known flu agents such as Tamiflu and Relenza could therefore also be used for inflammatory intestinal diseases, which opens up new therapeutic possibilities,” says Hennet. #  www.uzh.ch

FIC Proteins Send Bacteria Into Hibernation

Toxin-antitoxin

systems are ubiquitous in the bacterial world. The toxins usually inhibit protein synthesis or energy supply of the bacterium. Prof. Christoph Dehio’s research group at the Biozentrum, University of Basel, has uncovered a new mechanism of action of toxins from the group of FIC (filamentation induced by cAMP) proteins. Toxins among FIC proteins can be found in all domains of life. The researchers demonstrated that they act by altering cellular DNA. The FIC toxins modify two target proteins, called topoisomerases, which give the bacterial DNA its characteristic twisted shape and monitor its spatial structure. Topoisomerase activity is completely shut down by the toxins: “One can imagine as if FIC toxins pull the plug on topoisomerases,” explains Alexander Harms, first author and Fellowships for Excellence fellow at the Biozentrum. This rapidly leads to massive changes in the topology of cellular DNA, sending the bacteria into a kind of sleep state. In this state, the bacteria are protected from the action of the toxins or antibiotics, respectively, in order to persevere until environmental conditions get better. Until now, research has mainly focused on FIC proteins which are injected as virulence factors by pathogenic bacteria into host cells. In their study, the scientists led by Dehio demonstrated for the first time a biological function of evolutionarily more ancestral FIC proteins, which still act within bacterial cells. This discovery could help to understand how pathogens and their tools arise in evolution. #  www.unibas.ch

4

Lifesciences plus 02 I 2015


SWISS RESEARCH I News

How lipids are flipped

Biological membranes

have a fundamental role in separating the interior of cells from the extracellular space. They consist of a lipid bilayer with numerous other components embedded. Various vital processes, such as the transport of substances occur at membranes. The transport of phospholipids and lipid-linked oligosaccharides (LLO) is particularly difficult to achieve due to the bipolar nature of the lipid bilayer – hydrophobic interior, hydrophilic surface. This is why flippases are required to transport lipids from one side of the membrane to the other, essentially flipping their orientation. Flippases have important roles in maintaining the asymmetry of cellular membranes. They are also essential for transporting LLOs, which are transferred onto acceptor proteins during N-linked protein glycosylation. Three research teams from ETH Zurich and the University of Bern, led by ETH professor Kaspar Locher, have now determined the structure of one PglK flippase from the bacterium Campylobacter jejuni. The study has been published in the journal Nature. The researchers show that PglK consists of two identical subunits that move like a pair of scissors when energy (ATP) is consumed. Similar to a credit card reader, the oligosaccharide moiety of the lipid-linked oligosaccharide is then pulled into a hydrophilic channel within the flippase. The hydrophobic, lipidic tail of the LLO molecule remains exposed to the lipidic membrane, causing the LLO to change its orientation so that the sugar part ends up on the outside of the membrane. The flippase itself does not change its orientation during translocation reaction and therefore acts as a catalyst. There are diseases associated with mutations in a human flippase, explains ETH Professor Markus Aebi. Examples of this include the development and maturation of the central nervous system. Whether the newly acquired knowledge of the bacterial flippase PglK leads to novel therapeutic approaches is unclear at present. However, flippases already form an essential part of biotechnological systems that are used to generate glycoproteins designed for various uses in diagnostics and potential therapeutic agents. #  www.ethz.ch

Putting a stop to energy loss.

Proof efficiency! LLO synthesis and N-glycosylation in Campylobacter jejuni at a glance: The LLO’s lipidic tail is shown in purple. A polypeptide chain is shown in blue. PglH (red) is the glycosyltransferace that couples the oligosaccharide to the polypeptide chain (Picture: ETHZ)

FIC toxins modify the spatial structure of the DNA (blue) of bacteria (red: cell membrane). (Picture: University of Basel, Biozentrum)

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SWISS RESEARCH

CANCER THERAPY

Can Drug Resistance be Overcome? Treatment-resistant tumours are the most common cause of death for patients with metastatic cancer. An international research team led by Professor Sven Rottenberg from the University of Berne discovered how certain types of cancer build up resistance. The research results are expected to be very useful for improving chemotherapies of breast and ovarian cancer.

CLAUDIA BORCHARD-TUCH

M

edical scientists now know a lot about cancer — much more than they did ten years ago. About 500 genes

have been implicated in the disease so far, and the list is growing. There are also about 100 approved cancer drugs that specifically target mutations in those genes1 [Figure 1]. Everyone has BRCA (BReast CAncer)1 and BRCA2 genes. They are involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double-strand breaks – a process called homologous recombination (HR). Specific

inherited

mutations

in

BRCA1 and BRCA2 increase the risk of breast and ovarian cancer, and they have been associated with increased risks of several additional types of cancer. In cells with BRCA mutation, DNA is more frequently repaired by single strand break repair. This repair is accomplished by base excision repair (BER), nucleic acid excision repair (NER) or mismatch repair (MMR). BER is important for removing damaged bases by a DNA glycosylase and it is involved in the

6

Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I C a n c e r Th e r a p y

01

iStock

02

BRCA mutations are inherited in a genetically dominant

Poly (ADP-ribose) polymerase 1.

fashion, from either parent. (Pictures: Wikimedia)

03

Skeletal formula of olaparib.

04

Structure of protein encoded by REV7.

repair of the damage induced by radia-

tates the production of single strand

liferating cells. A major breakthrough

tion and alkylating agents.

break repair components. PARP1 ac-

came out in 2005 when the sensitivity of

MMR recognizes and corrects mis-

counts for more than 90 % of ADP-ribo-

BRCA1- and BRCA2-deficient cell lines

matched bases that can result from DNA

sylation in cells, while PARP2 is only re-

toward PARP inhibitors was demon-

replication and recombination. NER re-

sponsible for 15 % of the cell’s PARP2

strated3.

moves short single-stranded DNA seg-

production .

PARP inhibition in cells with BRCA

3

ments around the lesion and repairs mu-

PARP inhibitors first emerged 30

mutation leads to apoptosis: The loss of

tations resulting from UV light and hy-

years ago as potential anticancer drugs,

PARP (thus single strand break repair)

drocarbons .

showing an exquisite cytotoxicity in pro-

and BRCA (HR) is synthetically lethal. 

2

An Important Therapeutic Option: PARP Inhibition

Nonetheless, one has to ask whether

Single strand break repair requires Poly(ADP-ribose) polymerase (PARP) [Figure 2]. Seventeen structurally similar proteins compose the PARP family2. Particularly, research on PARP enzymes

overcoming drug resistance by chasing

mutation after mutation is a sustainable

as targets for cancer treatment has fo-

model for all types of drug resistance.

cused on PARP1 and PARP2. They catalyze poly(ADP-ribosyl)ation of proteins, using NAD+ as substrate, which facili-

Lifesciences plus 02 I 2015

7


S W I S S R E S E A R C H I C a n c e r Th e r a p y

Therefore, PARP inhibition seems to be a promising approach in the development of cancer treatment. In this con-

text, it is an advantage that BRCA-mu-

Specific inherited mutations in BRCA1 and BRCA2 increase the risk of breast and ovarian cancer. PARP inhibition in cells with BRCA mutation leads to apoptosis.

tations mostly appear in tumour cells. So there are fewer toxic side effects

Genetic reversion of BRCA-inactivating mutations

in comparison to conventional cancer therapy.

can be an underlying mechanism of drug resistance.

Different clinical trials demonstrated the efficacy of this approach. The orally active PARP inhibitor Olaparib [Figure 3] showed clinical benefit in BRCA1- or BRCA2-mutant tumours in-

that the loss of the gene re-establishes

Overcoming Drug Resistance

cluding ovarian, breast, and prostate

CtIP-dependent end-resection of double

Rottenberg’s research highlights the

types of cancer. Studies with other PARP

strand break in BRCA1-deficient cells.

potential of personalized medicine. Fig-

inhibitors such as Veliparib were also

The CtIP protein, which interacts with

uring out what mutation causes a drug

successful.

the C-terminal binding protein, plays an

resistance and designing a substance to

important role in HR repair through reg-

target it, is a technological triumph.

ulation of DNA end resection. CtIP is re-

Nonetheless, one has to ask whether

As PARP inhibitors have the potential

cruited to sites of DNA damage, where it

overcoming drug resistance by chasing

to act as therapeutic drugs against can-

associates with BRCA1 and the Mre11-

mutation after mutation is a sustainable

cer, a major challenge should be ad-

Rad50-Nbs1 (MRN) complex to promote

model for all types of drug resistance.

dressed: Drug resistance can occur by

HR-mediated DSB repair5. In this way tu-

To conquer resistance, researchers will

HR restoration. Genetic reversion of

mour cells with loss of REV7 become re-

need to answer some basic scientific

BRCA-inactivating mutations can be

sistant to therapy.

questions.

Drug Resistance

the underlying mechanism of drug re-

Rottenberg and his team developed

The tumour can be seen as an eco-

sistance, but this does not explain re-

an alternative treatment for this thera-

system, a mixture of cells that are con-

sistance in all cases4.

py-resistant form of cancer. The scien-

tinuously mutating. Treating physicians

In particular, little has been known

tists blocked the enzyme Ataxia Telangi-

put into that mix a very strong selective

about BRCA-independent restoration of

ectasia Mutated (ATM). ATM is a serine/

pressure, which is the drug. At that

HR. Recently, a research team around

threonine protein kinase that is recruit-

point it becomes survival of the fittest.

Professor Sven Rottenberg from the Uni-

ed and activated by DNA double-strand

Many cells die; others use a combina-

versity of Berne developed a BRCA-inde-

breaks5. It can be assumed that ATM ac-

tion of strategies to survive and thrive.

pendent

breakthrough

tivity is required for an early step in

These may include producing protein

brings important insights which facilitate

DNA repair, leading to ATR (a complex

pumps that flush the drug out, increas-

drug development in cases of therapy re-

of ataxia telangiectasia mutated and

ing the rate of DNA repair or using an

sistance.

Rad3 related) activation. Activation of

alternative molecular pathway to re-

DSB processing requires phosphoryla-

store whatever function the drug blocks.

tion of CtIP by ATR6.

Targeted drugs contribute to the genetic

method. This

The research team showed that loss of a special gene – called REV7 or MAD2L2

Based on results of successfully con-

complexity: The therapies themselves

[Figure 4] – in mouse and human cell

cluded studies in mice, Rottenberg and

can be driving tumours to become more

lines leads to HR restoration and PARP

his team are currently developing new

heterogeneous1.

inhibitor resistance. Normally, the gene

ATM inhibitors for clinical studies. Rot-

A better understanding of the under-

blocks repair in BRCA1-deficient cells.

tenberg expects initial clinical success

lying genetic diversity of tumour cells

Rottenberg and his team demonstrated

in four to five years.

may help researchers to work out how to tackle drug resistance. Some researchers are therefore exploiting ever-faster and

cheaper DNA-sequencing technologies.

Using a combination of drugs can

Other researchers are now trying to look at tumour evolution in patients. Biopsies

reduce a tumour’s options.

are taken from multiple spots within tumours both before and after treatment, then analyzed by sequencing the parts of the tumour genomes that code for

8

Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I C a n c e r Th e r a p y

proteins.

Comparing

these

biopsies

keep HIV in check. Like cancer, HIV has

should identify which mutations are as-

tremendous

sociated with drug resistance. These

evolves rapidly, but the right cocktail of

genetic

diversity

and

kinds of studies can be used to predict

drugs has transformed HIV infection

tumour evolution without having to do

from a death sentence for many into a

repeated sequencing studies to get fu-

manageable, long-term condition. It is

ture patients on the right therapies1.

possible that cancer can be tackled in a similar way1. 

Some researchers caution that genetics will provide only part of the picture of tumour heterogeneity and drug resistance. Variations in how tumours use

REFERENCES

these genes — the way they are regulated and expressed — also enable tu-

1

Bourzac K. Biology: Three known unknowns. Nature. 2014;509(7502):S69–71.

mours to develop drug resistance .

2

Toss A, et al. Molecular Mechanisms of PARP Inhibitors in BRCA-related Ovarian Cancer. J Cancer Sci Ther.

ing the way that tumours evolve in the

3

Yelamos J, et al. PARP-1 and PARP-2: New players in tumour development. Am J Cancer Res 2011;1(3):328–46.

face of chemotherapy, researchers are

4

Xu G, et al. REV7 counteracts DNA double-strand break resection and affects PARP inhibition. Nature.

1

But even without fully understand-

2003; 5:409–16.

coming up with ways to overcome re-

2015;521(7553):541–4.

sistance. Using a combination of drugs

5

can reduce a tumour’s options. Here,

Doublestranded Breaks. J Biol Chem. 2012;287(25):21471–80.

scientists take inspiration from the suc-

6

cess of the antiretroviral cocktails that

Forums

Posters

Courses, Workshops & Awards

Peterson SE, et al. Activation of DSB processing requires phosphorylation of CtIP by ATR. Mol Cell. 2013;49(4):657–67.

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Wang H, et al. CtIP Protein Dimerization Is Critical for Its Recruitment to Chromosomal DNA


SWISS RESEARCH

MEDICAL TECHNOLOGY

Personalized Medical Technology – Innovate for Health

Switzerland is famous for its excellent research in the field of life science. But how do things stand regarding innovations in medical technologies made in Switzerland? Unfortunately, possible applications often hardly make their way into the market in Switzerland and beyond. But nonetheless, there is a lot of innovative potential regarding personalized medicine in the discipline of medical technology.

SONJA BICHSEL-KÄSER

T

oday, about half of the industrial production and services in the Swiss economy is related to health. Hence, the recent growth

in medical device technology as a part of the health pillar is an important contributor to the Swiss Economy. The excellence of Swiss Science builds the basis for the strength of the health-related products on the market. Therefore, the label “Swiss made” is an asset for the Swiss industries. Thanks to this concept, Switzerland remains an attractive location for business. The more knowledge and capabilities rest in the country, the more success for research and development as well as for innovative technologies are guaranteed. But is it really that simple? On a symposium entitled “Swiss made in medical technologies and life sciences,” organized by i-net innovation networks and CSEM, experts discussed the poten-

Implants for hip, knee or shoulders are

tial and the obstacles regarding innova-

part of medical technology. Although

tion in medical technologies. There are

conventional implants vary in size, their

indeed a lot of success stories of medical

proportions are identical, which can

technologies made in Switzerland, most

misfit in some patients. (Picture: iStock)

of them are part of the trend to person-

Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I M e d i c a l Te c h n o l o g y

“Young scientists and surgeons even

alized medicine. But there are, of course, also some obstacles to overcome.

The company Symbios Orthopédie SA is an example for both success and

do not know how to bring an idea to market. They have to be encouraged.”

limitation. Symbios is producing tailored alternatives to conventional hip

HANS-FLORIAN ZEILHOFER. MD, University Hospital Basel

and knee implants. Though conventional prostheses are available at different sizes, they all have the same shape. But in some patients standard implants simply do not fit. Even after a successful

and obstacles in medical technology de-

ics,

Imaging

Technologies,

Material

surgery, the patients may still suffer

velopment. “Innovation has to be sup-

Sciences as well as Medical Science are

from unsatisfactory mobility or pain.

ported by small and medium-sized en-

involved in the development of new

Regarding hip implants, this is often

terprises,” he agrees with Plé, “big com-

technical equipment and techniques.

due to the individually different angles

panies usually see the risks before the

Therefore good networking is neces-

between the femur and acetabular cup.

potential.” But according to Zeilhofer

sary in order to join forces and create

The solution of Symbios is therefore the

the problem of sluggish development

new tools.

custom-made implant. By means of

of new medical technologies also lies

The actual trends in medical tech-

scanning, pelvis or knee of the patient is

within the medical research community.

nology innovations are intelligent sen-

reconstructed in 3D. Thus, based on a

“Many good ideas remain in the drawer,”

sors and ubiquitous health care as a

model the prosthesis can be tailored to

he regrets. “Young scientists and sur-

part of personalized medicine. Ubiqui-

fit the real joint. The company has been

geons even do not know how to bring an

tous healthcare is an emerging area of

founded 15 years ago. Symbios exports

idea to market. They have to be encour-

technology that uses a large number of

implants throughout Europe and the

aged.” This is where investors like i-net

patient sensors and actuators to moni-

success rate of custom-made prostheses

or CSEM jump in. They fill the gap be-

tor and improve patients’ physical and

for people under the age of 50 is around

tween research and transfer to the in-

mental condition. Tiny sensors gather

85 % which is 15 % more than is reached

dustry for further trials and commer-

data on almost any physiological char-

in conventional surgery.

cialisation of a potential product. “An-

acteristic that can be used to diagnose

The drawback of personalized im-

other obstacle for innovations in health

health problems. There are many ideas

plants is – as one might imagine – the

care technologiy is the hesitant posi-

and projects for intelligent sensor, such

costs. Health insurance companies do

tions of surgeons and physicians being

as sensors integrated in textiles like

not pay for the better fitting implants,

sceptical

technologies.

T-shirts, to measure transpiration or

which in turn are cost-intensive and

Since for new inventions, little experi-

other substances in the sweat. Bio-

laborious.

ence is available.”

chemical sensors can detect ions, gluta-

Jean Plé, CEO of Symbios, regrets

towards

new

mate, glucose, pH or amylase, which are

that healthcare is not able to support

Small Sensors Embedded in a Garment

the patients. But it is not only the insur-

According to Fabian Käser, Manager

diagnostics. Data transfer via Bluetooth

ances. “Financing questions arise also

Medtech at i-net innovation networks,

would then generate further access to

earlier, during development of a new

Switzerland is still on top of the world-

the data. Also monitoring of the heart by

technology. For start-up companies it is

wide charts regarding innovation. De-

ECG without gel patches and wireless

extremely difficult to find investors,

velopment in medical technologies is

could turn from a vision to a project in

since big companies are mainly inter-

usually interdisciplinary. Bioinformat-

the near future. 

often important parameters in clinical

ested in production and selling of a product,” he states. The true potential of an innovative idea does not matter too

In some patients

much when it comes to financing. “Unfortunately, financiers do not under-

stand orthopaedics.” Also Hans-Florian Zeilhofer MD,

simply do not fit.

Head of the Department for Oral and Maxillofacial Surgery at the University Hospital Basel, knows the difficulties

Lifesciences plus 02 I 2015

standard implants

11


S W I S S R E S E A R C H I M e d i c a l Te c h n o l o g y

through both, monitoring platform and real-time decision support system. In particular, the rapid development of big data, cloud computing, social networks, and smart phone further speeds up the applications of sensor networks for ubiquitous

healthcare

services. The

ubiquitous healthcare can be realized by integrating the sensing, communications, and computing together. Carmelo Bisognano, Head of Strategy at the Inartis Association, points out that in the future, patient John Doe does not exist anymore: Patient treatment by doctor appointment, drug prescriptions and final check-up at the doctor’s offices will no longer be the typical way. Visiting the doctor will happen virtually. “Digitalised and personalised healthcare will be part Ubiquitous Healthcare with sensor,

of everyday life in the future,” he is

cloud computing and smart phone

convinced. In the field of ubiquitous

technology will probably soon replace

healthcare,

conventional doctor’s appointments.

about the patient will be gathered and

(Picture: iStock)

the correct molecular diagnostic and

biomolecular

information

treatment can then be applied. Data generation, diagnosing and treatment will happen off-medical office and laboratory. However, there are also numerA story of success tells the company

ous challenges including the develop-

“Swisstom.” The company developed

ment of protocols, interfaces, platforms,

and manufactures a lung function mon-

and applications of sensor networks for

itor. This tool enables life-saving treat-

Microfluidic Chips offer new insight in tumour

ubiquitous healthcare. There is a strong

ments for patients in intensive care and

research by better mimicking of the tumour

need to develop advanced methods and

during general anaesthesia. Unlike tra-

micro-environment in comparison to conventional

algorithms to deal with questions such

ditional tomography, Swisstom’s bed-

tissue culture. (Picture: Wikimedia)

as energy optimization, harvesting, data

side imaging is based on a non-radiat-

fusion, decision support, data security

ing principle: Electrical Impedance To-

and privacy, system integrations, and

mography (EIT). The technology bases

smart system design. Again, knowledge

on weak alternating currents. They are

in all the relevant research fields from

applied to the body and travel along the

parable devices can show such regional

medicine to informatics has to be linked

paths of least resistance thereby creat-

organ function continuously and in

in order to create integrated healthcare.

ing electric potentials at the body sur-

real-time at the patient’s bedside.

face. These potentials are being meas-

Cancer Therapy from Chip to Patient

ured continuously by electrodes and

Big Data and Ubiquitous Healthcare

Daniel Zwahlen, medical superinten-

turned into tomographic images of lung

A challenge to address in the future will

dent at the Institute for Radiotherapy

function.

be the handling of the huge amount of

and Oncology of the Kantonsspital

The lung function monitor is used to

data. Wireless sensor networks can col-

Graubünden (KSGR), knows the impor-

continuously show lung function of ICU

lect physiological and environmental in-

tance of networking among the scientif-

patients undergoing mechanical venti-

formation of patients. The data is send to

ic disciplines. Thanks to a collaboration

lation. The patients simply wear a sen-

platforms or interfaces to trigger further

between KSGR and CSEM it is possible

sor belt with integrated active elec-

actions – either by alerting a surveil-

to gain insight into the impact of differ-

trodes to measure the respiration in dif-

lance system or by initiating implanted

ent therapies for bladder cancer from a

ferent lung regions, constantly convert-

actuators, which release pharmaceuti-

3D model.

ing the generated data into continuous

cals directly into tissue or bloodstream.

Worldwide, bladder cancer ranks

images. This gives insight into individu-

This enables high quality and ubiqui-

the fifth place of the most frequent

al personal conditions. To date, no com-

tous healthcare services to the people

cancer types. “75 % of the tumours are

12

Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I M e d i c a l Te c h n o l o g y

➜ “Digitalised and personalised healthcare will be part of everyday life in the future.” CARMELO BISOGNANO. Head of Strategy at the Inartis Association

“Financing questions arise early, during development

nique offers a better mimicking of the

similar projects worldwide. The next

tumour micro-environment in compari-

step in such studies is to find out which

son to conventional tissue culture. The

environment

dimensions of the chip are very similar

most therapy-relevant ex vivo responses.

to the native tissue and perfusion and

With that, the prediction of the most

delivery can be controlled. In the 3D cell

successful treatment for the patient will

culture, co-cultivation of tumour and

become even more precise.

give

Nevertheless, the technique looks

ent matrices, in order to fake natural tu-

promising. This will hopefully encour-

mour environment, can be tested. Cells

age more Swiss physicians and bio-

isolated from tissue after biopsy are

engineers to pursue the clinical direc-

tested for the tumour markers CD133 or

tion, which would help moving the field

ErbB-2. Cells harbouring these genes

of personalised medicine forward even

are thought to be possible cancer stem

faster, in order to make the latest tech-

cells and therefore are cultivated to

nologies accessible for the affected

form aggregates (spheroids) on a chip.

people. 

These spheroids can then be treated with radiation or chemicals. ORGANIZATIONS MENTIONED IN THIS ARTICLE

search is necessary for the approach to

JEAN PLÉ. CEO of Symbios SA

superficial and can be fought with

become gold standard in cancer treat-

• i-net Innovation Networks Switzerland: www.i-net.ch

ment. Fine tunings, like the optimiza-

• CSEM: www.csem.ch

tion of micro channels or studies with

• Swisstom AG: www.swisstom.com

more patient samples have to be carried

• Inartis Association: www.inartis.ch

out. This is already being done in many

• Symbios Orthopédie SA: www.symbios.ch

surgery, chemotherapy and immunotherapy,” Zwahlen states. The latter comprises instillation with BCG (Bacillus-Calmette-Guérin). The germs induce a local inflammation which activates

the

body’s

immune

reaction

against tumour cells. “Unfortunately, in bladder cancer recurrence rate is high, about 20–25 %, which is poor,” Zwahlen points out. In order to investigate the possibilities

of

personalized

cancer

therapies, the collaboration was started. Whilst KSGR brings in clinical expertise, knowledge and equipment for

Master of Science (MSc) in Life Sciences

radiotherapy and access to tumour tissue, CSEM provides know-how in mi-

Vertiefungen in Food and Beverage Innovation Pharmaceutical Biotechnology Chemistry for the Life Sciences Natural Resource Sciences

crofluidics technology and equipment of a cell culture laboratory. And this is how the two partners profit from synergies: Bladder stem cells and tumour tissue is harvested from patients. Tumour tissue can then

Info-Anlass in Wädenswil

be grown in vitro and cancer stem cells

Dienstag, 29. September 2015 18.00 Uhr, Campus Grüental, Wädenswil

(such as CK5, CK17, DC44 and 67LR) can be isolated. By flow cytomtery and genetic analysis, the cells and tissues are screened for biomarkers like p532, pRB and others. In a further approach of personal-

Zürcher Fachhochschule

ized medicine, a 3D tumour cell culture is grown by microfluidics. The tech-

Lifesciences plus 02 I 2015

the

non-tumour cells is possible and differ-

For sure, more experience and re-

of a new technology.”

combinations

13

www.zhaw.ch/lsfm/master-lifesciences


SWISS RESEARCH

HUMAN PHYSIOLOGY

Sound Asleep for Research During deep sleep our brains remain active, generating distinct signals that can be detected by electroencephalography (EEG). Sleep researchers who joined forces in cooperative projects of the Zurich Center for Integrative Human Physiology (ZIHP) relied on this method to examine the influence of genetic variations and intelligence on sleep and to obtain novel insights into brain development.

iStock

14

Lifesciences plus 02â&#x20AC;&#x2030;Iâ&#x20AC;&#x2030;2015


SWISS RESEARCH I Human Physiology

CHRISTINA GIGER

A

Children with a higher IQ might need

re our individual sleep characteristics genetically predetermined? Is our brain able to perform better if we sleep

less sleep because their brains not only work more efficiently but also regenerate faster.

more? Does an intelligent brain need more or less sleep? Can sleep even provide information about the current level of maturity of a child’s brain? To answer such questions of enormous social relevance, an interdisciplinary research approach is needed,

trodes that record potential differences

teristics. For a publication evolving from

involving a team of experts in both

on the scalp. These differences result

this ZIHP cooperative project, the ZIHP

basic and clinical research. The Zurich

from electrical signals of brain cells and

award was given last summer.

Center for Integrative Human Physiology

reflect the transmission and processing

(ZIHP) promotes this research ap-

of information in the brain. Thus, they

Intelligent Kids Need Less Sleep

proach by granting cooperative projects

are a robust, large-scale measure of ne-

Apart from such distinct genetic differ-

in which at least three ZIHP members

ocortical dynamics and closely related

ences, sleep demand also changes dur-

collaborate to generate a synergistic

to the state of the brain.

ing human development and aging. Chil-

value. The results of ZIHP-funded pro-

During deep non-rapid-eye-move-

dren need more sleep because their

jects in sleep research confirm the ben-

ment (NREM) sleep the reduced brain

brains are still developing. Nevertheless,

efit of this approach.

activity is represented by waves with low

not all kids need the same amount of

frequency (“slow waves”) in the EEG. It

sleep. ZIHP researchers were interested

is assumed that deep sleep contributes

to investigate whether children’s sleep

to the storage and formation of new

duration influences their cognitive per-

To find out how genes determine sleep

memories. Moreover, deep sleep is be-

formance. They studied eight to twelve

features, ZIHP researchers studied a

lieved to be an important recovery phase

year old school kids who had to fill in

gene variant of an enzyme that metabo-

for the brain. A shortage of sleep one

questionnaires, keep a sleep diary to-

lizes adenosine. Adenosine is important

night will be compensated by a deeper

gether with their parents and wore activ-

for cellular energy production, and in-

and longer sleep the following night.

ity monitors to measure their rest/activi-

Gene variant causes sleepiness and reduced vigilance

duces sleepiness by triggering sleep-pro-

The fact that the volunteers with the

moting brain regions. This gene variant is

gene variant sleep deeper and have

present in around 10 % of the Caucasian

longer periods of “slow wave sleep” sug-

population, rendering the enzyme less

gests that they may suffer more during

showed that children who needed less

effective so that adenosine is only partly

sleep deprivation. This suggestion was

sleep had a higher IQ. Particularly, they

metabolized.

supported by additional tests during

had a higher ability to solve new prob-

To test whether this small genetic

prolonged wakefulness. The activity of

lems independently of acquired knowl-

modification affects sleep, ZIHP re-

α-amylase – a proposed biomarker for

edge. Does this mean that we should keep

searchers kept volunteers awake for for-

sleepiness – was determined in saliva.

our children awake in order for them to

ty hours in a sleep laboratory. Interest-

The volunteers also had to rate their

become more intelligent? The research-

ingly, the volunteers carrying the muta-

sleepiness and mental state on ques-

ers clearly oppose such a suggestion.

tion slept deeper before and after the

tionnaires. All tests led to the same re-

Children with a higher IQ might need

sleep deprivation than the volunteers

sult: The subjects with the mutation

less sleep because their brains not only

with the normally functioning enzyme.

were sleepier and less attentive than

work more efficiently but also regenerate

the persons with a normally functioning

faster. If kids who need more sleep are

employed electroencephalography (EEG).

adenosine-degrading

These

forced to sleep less, this could even have

This method allows to examine various

observations show that genetic factors

negative consequences on their cognitive

features of sleep with the use of elec-

indeed have an impact on sleep charac-

performance and development. 

To quantify sleep depth, researchers

Lifesciences plus 02 I 2015

15

enzyme.

ty patterns. Additionally the children were performing an intelligence test. Counter-intuitively,

the

results


SWISS RESEARCH I Human Physiology

➜ Volunteers with a gene mutation were sleepier and less attentive than the persons with a normally functioning adenosine-degrading enzyme.

A topographical map of the active brain regions can give a picture of the current state of maturity of the brain.

EEG-Imaging of Brain Development

in attention-deficit/hyperactivity disor-

ZIHP researchers also estimated brain

der (ADHD). ZIHP researchers report-

development in children and adoles-

ed that several characteristics in the

cents by measuring the EEG during deep

EEG of ADHD-children differ from

sleep. Changes in brain activity in specif-

those of healthy peers. In a follow-up

ic regions are observed during matura-

project it will be determined to what ex-

tion or structural reorganisation. A topo-

tent the EEG together with other meas-

graphical map of the active brain regions

urements can be used as diagnostic

can therefore give a picture of the cur-

markers for ADHD.

rent state of maturity of the brain.

As a center of competence gener-

Employing this method, researchers

ously funded by a University Research

found that the brain development of

Priority Program of the UZH, the ZIHP

children and adolescents born preterm

is proud of these projects that have con-

might be altered compared to term born

tributed new insights and understand-

peers. Thirteen year old children born

ing in the fields of sleep research and

prior to the 32nd week of gestation and

human physiology, and of their transla-

with a birth weight of less than 1500

tion to the clinics. 

grams performed slightly worse in tests for planning and working memory than their term born peers. Also their average IQ was lower, however still within normal limits. AUTHOR

EEG for ADHD Classification EEG is also used in other fields of re-

Dr. Christina Giger. Scientific Coordinator at the Zurich

search, for example to improve the un-

Center for Integrative Human Physiology (ZIHP) of the

derstanding of the brain changes seen

University of Zurich

Brainwaves change during modulation of mind activity. (Picture: iStock)

Electroencephalography (EEG) is employed to quantify sleep depth: The method is used to examine various features of sleep with the use of electrodes that record potential differences on the scalp. (Source: ZIHP)

16

Lifesciences plus 02 I 2015


SWISS RESEARCH

TISSUE ENGINEERING

Periodontitis – The Insidious Disease Widespread and underestimated, periodontitis is a serious periodontal inflammation that damages our soft tissue and destroys teethsupporting bone, causing tooth loss and additionally increasing the risk of a heart attack or stroke. Credentis AG, the international dental health firm, is aiming to achieve complete tissue regeneration based on a matrix of self-assembling peptides. To this end, Credentis researchers are cooperating with ZHAW Wädenswil and the University of Zurich.

ELSBETH HEINZELMANN

A

ccording to the World Health Organization (WHO), 5 to 15 % of the global population suffers from advanced periodon-

SEM image of cleaned dentine surface. (Magnification: 2000 ×) (Photos: ZHAW Wädenswil)

titis. Since the condition is painless, it is often not taken seriously. Periodontitis is caused by certain bacteria and the local

for special cleaning and the removal of

specialized in the natural regeneration

inflammation they initiate. Although the

plaque and tartar deposits on the tooth

of bone and soft tissue in dentistry, he

bacteria are naturally present in the oral

and root surfaces. This helps the tissue

remembered a special peptide technol-

cavity, they may become dangerous un-

surrounding the tooth to heal and peri-

ogy that had been a main focus of study

der specific conditions and increase dra-

odontal pockets to shrink.

of scientists at the University of Leeds.

matically. This is the case when a layer of

As soon as periodontitis has devel-

Would it not be possible to use intelli-

bacteria and food debris – plaque – builds

oped, several painful techniques are ap-

gent peptides to regenerate mineralized

up and remains ignored, especially in in-

plied to regenerate the affected tissue.

tissue and to exploit this potential for

accessible sites like interdental spaces.

Manual operations, the application of

the treatment of caries? In January 2010

grafting materials and the use of enam-

he founded the start-up Credentis AG to

el matrix proteins are the current

translate his vision into reality.

A step ahead of the competition In the early phase of periodontitis,

standardized therapies.

First he had to convince investors by

known as gingivitis, there is no medical

When the biochemist Dominik A.

arranging the exclusive licensing of pa-

need for surgical treatment, but simply

Lysek worked in a leading company

tents for the worldwide application for

Lifesciences plus 02 I 2015

17


S W I S S R E S E A R C H I Ti s s u e E n g i n e e r i n g

teeth. Clinical studies were initiated and

SEM image of human periodontal

performed. In parallel, the crude tech-

fibroblasts growing on a bovine

nology was translated into a marketable

dentine surface. Cells attach to

product, “Curodont Repair.” This turned

this natural surface, but increased

out to be a gigantic technological leap.

tissue regeneration capacities are

Curodont Repair uses the natural remin-

expected in combination with the

eralization potential of teeth to cure ear-

peptide matrix. (Magnification: 800 ×)

ly caries lesions. The key component is a patented peptide that is present as a monomer when the product is dissolved.

works or scaffolds that can be used for

However, within the carious lesion the

biological processes such as mineraliza-

peptides self-assemble to form a large

tion of enamel, why should a natural

fibre network or matrix. When Curodont

process of tissue regeneration not be

Repair is applied onto a carious lesion

possible with the same procedure, pro-

the monomers diffuse through the pores

vided of course that external factors, e.g.

of the hypermineralized plate into the

bacterial growth, do not interfere with

subsurface lesions. “Within the subsur-

the regeneration? The periodontal liga-

face cavities they form the three-dimen-

ment is a very complex structure be-

sional matrix around which denovo crys-

tween the tooth cementum and the al-

tallization of calcium phosphate occurs,”

veolar bone. The matrix of the perio-

explains Dominik Lysek. “This process

dontal ligament is built up by periodon-

is equivalent to the natural mineraliza-

tal ligament fibroblasts (PDLF). In a

tion process that occurs during odon-

regenerative process, precursor cells

togenesis or the remineralization pro-

are recruited from the root and the sur-

cess which – for a healthy tooth – is in

rounding tissue to restore the defective

equilibrium with the demineralization

tissue. To enable the migration of the

process.“

cells,

Taking Inspiration from Nature

an

appropriate

scaffold

onto

which the PDLFs can attach and spread out must be present. The integration of

But meanwhile Dr. Lysek is already

regenerative peptide matrix basically

thinking of the next step: When intelli-

requires no additional surgical inter-

gently designed peptides have the abili-

vention, is easy to apply by the dentist

ty to form three-dimensional fibre net-

and directly applicable during or after

the cleansing process. The idea is obvious and is increasingly being studied by research groups in the UK, China, US and the Netherlands, as self-assembled

Bovine teeth are used for establishing the in vitro system

peptide nanofibres exhibit adequate

as the dentine is similar to human dentine.

characteristics of a matrix for tissue regeneration processes. Already a series of synthetic peptides have proved to be

A 3D model of the periodontal ligament is established in a cell culture plate. Cells of interest are integrated in a collagen-based gel and migrate onto a natural dentin surface that can be coated with the investigated peptide matrix.

18

Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I Ti s s u e E n g i n e e r i n g

Dominik A. Lysek, founder and CEO (right) with a sound knowledge in chemistry, dental/oral biology and the development of medical devices, and CTO Michael Hug, responsible for production, supply chain management and quality control within Credentis. (Photo: Credentis)

Dr. Stephanie Mathes, senior scientist and scientific project leader at the Zurich University of Applied Sciences (ZHAW) Wädenswil.

capable of supporting tissue regenera-

proaches.” Also on board is the Center

tides are suitable for the regeneration

tion, but none has yet reached the stage

of Dental Medicine (CDM) of the Uni-

of the periodontal apparatus. Another

of clinical application.

versity

Professor

important issue is to investigate the fea-

Ronald E. Jung, a specialist in recon-

sibility of integrating an antimicrobial

structive dentistry, is the clinical advisor

substance in order to prevent microbial

for the project.

growth, which is the key factor for peri-

A Network of Strong Partners To develop this novel approach and join

of

Zurich, where

forces with effective partners, Credentis

A key issue of the project is the de-

odontitis progression. By applying an

AG set up a CTI project (Commission

sign of a guiding scaffold, i.e. how to

antibiotic drug-releasing matrix over a

for Technology and Innovation) with the

find a way to bring together short syn-

period of 4 to 5 days the process of tis-

group

Ursula

thetic peptides to form a matrix. Cre-

sue regeneration is supported with a

Graf-Hausner, research manager for

dentis has obtained the exclusive li-

high degree of reliability. The close co-

culture technology and tissue engineer-

cence to market this type of peptides.

operation with an experienced special-

ing, and Dr. Stephanie Mathes, senior

Within the context of the project the

ist in hard and soft tissue regeneration

scientist and scientific project leader,

scientists want to identify which pep-

is therefore crucial for success. 

headed

by

Professor

both at the Zurich University of Applied Sciences (ZHAW) Wädenswil. “Over the last few years, they have been exploring tissue from 3D cell culture models and conducting active research in the dental sector,” declares CTO Michael Hug, who has himself worked in the field of bone and tissue regeneration. “They understand the special requirements of industry and develop highly practical ap-

Lifesciences plus 02 I 2015

5 to 15 % of the global population suffers from advanced painful periodontitis, which is treated by manual operations, the application of grafting materials and the use of enamel matrix proteins. 19


S W I S S R E S E A R C H I Ti s s u e E n g i n e e r i n g

While Credentis characterizes the basic

gated self-assembling peptide is the

The Core Factor – Small Peptides

RADA16 polypeptide. The nanofibre

components and the resulting matrix in terms of degradability and mechanical properties, while bearing in mind the final product, the ZHAW researchers evaluate different peptides with respect to their ability to support special characteristics of the tissue regeneration in vitro on the basis of a 3D periodontal ligament model. Their expertise lies specifically in the realization of this 3D model and the in vitro characterization of differentiation processes of specific tissue types. As soon as a preferred pep-

scaffold produced at the end of the RA-

Rather than a repair of the periodontal ligament, the project partners want to obtain a complete regeneration of the tissue based on a matrix of self-assembling peptides.

DA16 assembly process promotes bone, cartilage and neural regeneration and angiogenesis, either on its own or functionalized by direct coupling to short biologically active motifs. Although preliminary in vitro studies with periodontal

ligament

cells

indicated

that

RGD-functionalized RADA16 peptides were usable (Kumada et al., PLOS, 2010), this approach did not make its way from the bench to the bedside. As Credentis has the license to mer-

tide emerges from the in vitro tests, Pro-

chandise multiple small peptides from a

fessor Ronald Jung and his group at

library, the search for the most promis-

the CDM will verify the peptide in a re-

ing candidate is under way. This tunable

liable animal model and analyze it in vi-

Med, Jun 2015) published their data

system of synthetic peptides provides

vo for effectiveness.

concerning the regeneration of neu-

multiple opportunities especially in the

As mentioned before, the idea to use

ronal tissue using silk fibroin modified

context of regeneration of such a com-

small synthetic peptides for purposes of

by an Ikav peptide. Comparison with the

plex tissue as the periodontal ligament.

tissue regeneration is promising, as has

control group revealed increased cell

To evaluate the best candidate in an ef-

been revealed by various studies. Just

viability and an enhanced neuronal dif-

fective way, a predictive in vitro model

recently Sun et al. (J Tissue Eng Regen

ferentiation capability. A widely investi-

based on natural dentin is needed. “It is

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Lifesciences plus 02 I 2015


S W I S S R E S E A R C H I Ti s s u e E n g i n e e r i n g

absolutely essential to determine the

One thing is clear: Credentis and its

when the chemistry is right – as in our

main read-out parameters and applica-

project partners will need to have con-

case – one and one does not make two,

tion conditions in close coordination

siderable staying power until the finale

but three!” 

with the surgeons. As a model can never

is reached in 2017. But the project goal

reflect all the parameters of a given

has been clearly formulated: Rather

problem, we have to focus on the key

than a repair of the periodontal liga-

outcomes to be achieved in vivo and de-

ment, the project partners want to

sign the system appropriately so as to

obtain a complete regeneration of the

be successful,” explains Dr. Stephanie

tissue. This translates into clear advan-

OLTEN MEETING 2015 –

Mathes.

tages for the patient: As the growth of

WHERE THE EXPERTS MEET!

epithelial cell is inhibited, a short junc-

A Hard but Promising Road

tional epithelium can be formed. This

On 18 November at Hotel Arte in Olten the biotech branch

By the end of the year the 3D model

will result in a new acellular fibre ce-

shares ideas on regenerative medicine with latest findings

should be established. In summer 2016

mentum on the exposed root surface.

in antibiotics research and bioprinting.

the peptides from the library will be

New periodontal ligament will develop

Information: www.biotechnet.ch

evaluated with the model and the

with functional fibre orientation. Last

Professor Franz Baumberger,

characterization of the peptides will

but not least, we will observe a new al-

franz.baumberger@biotechnet.ch, +41 (0)78 666 0175

have

four

veolar bone extending to 2 mm below

months later the differentiation profile

the cement-enamel junction. CTO Mi-

will be analyzed within the new matrix,

chael Hug goes to the heart of the mat-

as will the degradation kinetics of the

ter: “At Credentis we are convinced that

matrix itself. The studies in this area

we have the best partners to master the

• www.credentis.com

will be concluded in 2017, and the data

challenges we are facing in this CTI

• www.icbc.zhaw.ch/de/science/icbc/fachstellen/

collected will be employed in the prod-

project: All of us are experts with com-

uct marketing.

petence, experience and creativity. And

been

completed.

About

ORGANIZATIONS MENTIONED IN THIS ARTICLE

mikro-und-zellbiologie.html • www.zzm.uzh.ch/research_en.html

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SWISS RESEARCH

SWISS START-U Ps

DRUG RESEARCH & DEVELOPMENT

Fighting Tuberculosis by Restoring Ethionamide’s Antibiotic Power Selective Inhibitors Researchers from Lille in France, GlaxoSmithKline in Tres Cantos, Spain, and BioVersys AG, a biopharmaceutical company based in Basel, are cooperating in developing a preclinical candidate able to restore ethionamide’s antibiotic power against Mycobacterium tuberculosis. BioVersys is also pursuing adjuvant strategies for potentiation of other antibiotics. The objective is to overcome antimicrobial resistance. BEATE PEISELER-SUTTER

S

ics,” summarizes BioVersys’ CEO and

important second-line drug has been

ynthetic biology is about com-

co-founder Marc Gitzinger. Suitable tar-

developed in the late 1950s and is still

bining genetic information of

gets may be regulatory pathways con-

essential when treating patients suffer-

different origins to new func-

trolling antibiotic induced thickening of

ing

tioning biologic cycle systems.

the cell wall or biofilm production.

drug-resistant

from

an

infection (MDR)

with

multi-

tuberculosis

Self-developed or purchasable nucleic

The company’s researchers gain im-

pathogen Mycobacterium tuberculosis.

acids sequences, so-called BioBricks,

portant insight into such kind of resist-

MDR-TB treatment extends about two

are assembled in a multi-species way

ance mechanisms by doing knockout

years and shows severe side effects due

and transferred to host cells. The exten-

experiments on laboratory strains and

to the limited efficiency of ETH bal-

sive possibilities of use and application

studying patients’ bacterial isolates of

anced by high dosages. The team of mi-

in this young field of research have

which BioVersys holds a large collec-

crobiologist Alain Baulard, Research

given birth to several new business ide-

tion.

Director at the Center of Infection and

as and start-up companies. One of them

Immunity of the French Pasteur Insti-

company spin-off from the “Department

Understanding Resistance Mechanisms in Tuberculosis

of Biosystems Science and Engineering”

Currently, work is carried out on three

abolic activation of antibacterial pro-

(D-BSSE) of the “Swiss Federal Institute

projects,

in

drugs, elucidated the transcriptional

of Technology” in Zurich (ETHZ). Bio-

Gram-negative as well as Gram-posi-

mechanism by which M. tuberculosis

Versys was founded in September 2008

tive bacteria; the most advanced one is

controls its own sensitivity to ethion-

and is based in the “Technologie Park

in the field of tuberculosis (TB). Several

amide (ETH).

Basel,” where it rents offices and labo-

antibiotics used in TB treatment are so

ratory space. “We focus on transcrip-

called

substances

ciency is that the transcriptional re-

tional regulators, which we target with

need to be bioactivated by enzymes of

pressor EthR negatively regulates the

small molecules in order to inhibit re-

the pathogen before exerting antimicro-

expression of the gene coding for the

sistance mechanisms that are switched-

bial activity. One such antitubercular

enzyme EthA, a Flavin-containing bac-

on in the presence of specific antibiot-

pro-drug is ethionamide (ETH). This

terial mono-oxygenase, which catalyzes

is BioVersys AG, a biopharmaceutical

addressing

resistance

pro-drugs. These

22

tute in Lille, with expertise and specializing in mechanisms of action and met-

“The reason for ETH’s limited effi-

Lifesciences plus 02 I 2015


➜ “We focus on transcriptional regulators, which we target with small molecules in order to inhibit resistance Dr. Marc Gitzinger, CEO of BioVersys (Picture: Peiseler)

mechanisms that are switched-on in the presence of specific antibiotics.” DR. MARC GITZINGER. CEO BioVersys

The next milestone will be the nomination of a substance candidate for development from out of the five different substance classes, which the consortium holds in hands thanks to joining their forces.

Lifesciences plus 02 I 2015

the required activation of ETH,” ex-

and was decisive for company founda-

plains Gitzinger. Transcriptional re-

tion. It enabled the discovery of tran-

pressors as well as activators mediate

scriptional regulator inhibitory com-

transcription-inhibiting or -activating

pounds differing from the structure of

signals via specific gene sequences

2-phenylethyl-butyrate and largely su-

called operators. EthR suppresses the

perior in activity. “Our screening and

biosynthesis of EthA via the operator

medicinal

OethR. As a result, EthA is produced in

yielded new compounds that fully reac-

insufficient amounts: an intrinsic resist-

tivate ETH sensitivity of all tested clini-

ance mechanism, protecting M. tubercu-

cal MDR and extensively drug-resistant

losis from ETH’s toxic effect.

(XDR) TB isolates in vitro at very low

Based on this know-how, Gitzinger, together with former colleagues from

chemistry

activities

have

concentrations,” says Gitzinger.

the research group of Martin Fusseneg-

Working in the Same Direction

ger, Professor at D-BSSE and co-found-

At the same time, Benoîit Déprez and

er of BioVersys, assembled a synthetic

colleagues from the 2007-founded “Pole

network, which is displaying the mech-

of Interdisciplinary Research on Drugs”

anism of ETH resistance. It was linked

(PRIM: Pôle de Recherche Inter-disci-

to a quantitative reporter gene expres-

plinaire sur le Médicament) in Lille

sion readout and transferred to mam-

started searching for EthR inhibitors

malian

cellular

that would boost antituberculous activi-

system was used to screen for com-

ty of ETH. The French researchers de-

pounds preventing EthR from binding

veloped a chemical screening system,

to OethR. It turned out that the nontox-

indicating compounds that disrupt the

ic flavor 2-phenylethyl-butyrate abol-

interaction of EthR with DNA. Co-crys-

ishes EthR’s repressor function inside

tallization of such molecules with EthR

human cells, in mice, and within M. tu-

brought additional information about

berculosis, where it triggers derepres-

the complex’s three-dimensional struc-

sion of ethA and increases the patho-

ture and led to the synthesis and opti-

gen’s sensitivity to ETH. The findings

mization of improved analogs. As well

were published in PNAS in July 2008.

as the team from BioVersys, the crew

The efficient screening system, devel-

from PRIM was able to develop active

oped during Gitzinger’s doctoral stud-

substances, which increase the potency

ies, set the starting point for Bioversys’

of ethionamide by a multiple: results

TRIC- technology (transcriptional reg-

that were published in Nature Medicine

ulator inhibitory compound) platform

in 2009. 

cells. The

23

artificial


SWISS RESEARCH I Drug Research & Development

Knocking out Bacterial Resistance Mechanisms

In January 2015,

Extensively drug-resistant

Nature Magazine

published a paper on the discovery of teixobactin, a natural compound

tuberculosis is a tuber-

isolated from the soil bacterium Eleftheria terrae, showing activity against Gram-positive bacteria such as Staphylococcus aureus

and Mycobacterium tuberculosis by binding to important cell wall forming precursor molecules. “It looks like a promising compound,

culosis form that is resistant to at least four of

but knowing the drug development risks, we need more early stage projects like this“, comments BioVersys CEO Marc Gitzinger Nature’s

the core anti-TB drugs.

rating of teixobactin as an “irresistible newcomer.” Gitzinger recalls that nature is a large pool for resistance genes. At least the organism, from which a natural compound has been isolated, is in possession of an intrinsic resistance mechanism. Besides acquiring antibiotic resistance through spontaneous mutations, bacteria can pick up resistance genes from other bacteria by horizontal gene transfer when

“When the French scientists and we no-

undergoing conjugation or by intervention of viruses (bacteriophages).

ticed that we were working in the same

Bacteria can also acquire naked DNA from their environment.

direction, contact was established with

Resistance genes can be accumulated in bacterial genomes over time

the doctor Alain Baulard, and the pro-

or at one fell swoop, leading to resistance against many different

fessors Nicolas Willand, and Benoît

families of antibiotics. It seems to be just a matter of time, until an

Déprez, all three leading scientists in

antibiotic loses its power given against resistant bacterial strains.

TB research and drug discovery from

This is the reason why BioVersys wants to explore alternative ways.

different institutions in Lille,“ tells

The Swiss company is closely looking on resistance mechanisms,

Gitzinger. The researchers agreed to

which could be inhibited by non-antibiotic compounds, following the

work together and started cooperation

example of Augmentin, a medicine that combines the β-lactamase

in 2011. In 2012, the “Société d’Ac-

inhibitor clavulanic acid with penicillin-group antibiotics. Clavulanic

célération du Transfert de Technologie”

acid its able to fight antibiotic resistance in bacteria that secrete

(SATT Nord), a center for technology

β-lactamase, which otherwise inactivates most penicillins.

transfer, took over all patents concern-

“The inhibition of enzymes like β-lactamase has been known for more

ing the cooperation that were formerly

than 20 years. We focus on more complex resistance mechanisms,

held by different institutions: the Pas-

which are tightly controlled by transcriptional regulators. Since their

teur Institute of Lille, the University of

constitutive expression would mean an unnecessary energetic cost

Lille, the National Center of Scientific

for the bacterium, such resistance mechanisms are up-regulated

Research (CNRS), and the National In-

only in the presence of antibiotics, which they turn against,” explains

stitute of Health and Medical Research

Gitzinger. Next to an inhibition of the EthR/OethR interplay in

(Inserm). “This smart move made col-

Mycobacterium tuberculosis in order to fight resistance to ethion-

laboration much easier. We currently

amide (see: Fighting tuberculosis by restoring ethionamide’s

share all rights on intellectual property

antibiotic powermain article), the expression of some efflux pumps,

while commercial rights are completely

may be another appropriate target. Such transport proteins are found

with BioVersys. All IP generating re-

in both Gram-positive and -negative bacteria as well as in eukaryotic

search falls under one same contract,”

organisms. They are involved in the extrusion of toxic substrates

explains the CEO of BioVersys. The col-

like antibiotics from within cells into the external environment.

laboration of the Swiss and French re-

Pumps may be specific for one substrate or may transport a range

searchers yielded new promising re-

of structurally dissimilar compounds. The latter are associated

sults, and in 2014, UK drug giant Glax-

with multiple drug resistance. BioVersys is investigating such

oSmithKline (GSK) joined the team.

resistance mechanisms. Together with the world-renowned antibiotics

The object is to develop a preclinical

expert David Livermore, Professor of Medical Microbiology at the

candidate against tuberculosis. Accord-

University of East Anglia and a member of BioVersys’ scientific

ing to Gitzinger, there are 14 people

advisory board, the Swiss scientists are also looking on transcrip-

working full-time on the project. All

tion-regulated pathways central crucial for cell wall thickening

work on absorption, distribution, me-

and biofilm production.

tabolism, and excretion of active com-

24

Lifesciences plus 02 I 2015


SWISS RESEARCH I Drug Research & Development

pounds as well as some in vivo experi-

tion of a substance candidate for de-

ments are conducted at GSK. while in

velopment from out of the five dif-

vitro and in vivo experiments with M.

ferent substance classes, which the

tuberculosis, requiring biological safety

consortium holds in hands thanks to

labs, and a main part of the medicinal

joining their forces. GSK is granted

chemistry activities are carried out in

the possibility to negotiate an over-

Lille. All other work is done at BioVer-

all license with BioVersys. 

www.schaefer-tec.com LS Instruments

sys in Basel. The Welcome Trust, who was already supporting GSK in order to push some early-stage projects focusing on

finding

treatments

for

NEW

diseases

largely affecting the developing world,

NanoLab 3D (DLS, very high concentrations)

co-funds the project, which is based on milestones. This means that further funding follows demonstrated progress. The next milestone will be the nomina-

NEW

Tuberculosis Remains a Life-Threatening Health Problem

Behind HIV infection,

DWS RheoLab (Micro-Rheology)

NanoAssemblr TM tuberculosis (TB) is on the second

place of deadly infectious diseases, caused by a single infectious agent. Nine million new TB cases were reported in 2013. In the same year, 1.5 million people died from the contagious disease, which is transmitted by infectious aerosol droplets. TB is most prevalent in Africa, India, China and Eastern Europe; infections with multidrug-resistant (MDR) and even extensively drug-resistant (XDR) Mycobacterium tuberculosis are on the rise. MDR-TB is when M. tuberculosis fails to respond to a combination of two of the four first line anti-TB drugs (rifampicin and isoniazid). Patients usually acquire drug resistant TB either as a result of spread of a drug resistant strain from another

Liposome nanoparticle production

CytoViva

TB sufferer or as a result of inappropriate or incomplete treatment. XDR-TB is a form of TB that is resistant to at least four of the core anti-TB drugs (rifampicin, isoniazid, fluoroquinolones and second-line injectable agents). In December 2012, the US Food and Drug Administration (FDA) approved bedaquiline, the first new anti-TB drug in 40 years. Bedaquiline affects the proton pump for ATP synthase and thereby thwarts the bacterial energy supply. Another new anti-TB drug, delamanid, received its first global approval at the end of 2013 from the European Medical Agency (EMA). Delamanid damages the pathogen’s cell wall. Both, bedaquiline and delamanid, are exclusively

Label-free nanoscale imaging

CytoSurge

reserved for the treatment of proven MDR- and XDR-TB. Anyway, despite these two new anti-TB drugs, the pipeline of high-quality treatments remains thin. “The existing price/volume business model for antibiotics is not working and is a key barrier to achieving more rapid progress on resistance,” argues Kevin Outterson, Professor at the University of Boston, in a recently published report on the broken market for antibiotic innovation. The authors recommend the authorities to continue “building on recent promising steps by boosting funding for basic research and development, surveillance, and antibiotic stewardship; targeting these initiatives to prior unmet needs; and reforming reimbursement to support effective antibiotic access and use rather than volume.”

Lifesciences plus 02 I 2015

25

Single cell manipulation

Schaefer-Tec AG

Badimatte 21 CH-3422 Kirchberg info@schaefer-tec.ch www.schaefer-tec.com


SWISS RESEARCH

PHYTOPHARMACEUTICALS

SWISS START-U Ps

Drugs from Hairy Roots BEATE PEISELER-SUTTER

Since 2007, the Swiss company ROOTec AG is doing R&D activities on the production of bioactive substances of plant origin. Natural compounds are eco-friendly produced by hairy root cultures, which are grown in in-house developed low cost mist bio-reactors for customers from the pharmaceutical, food, and cosmetic industries. Even though the technology is in place and experts see great potential in it, Swiss investors remain reluctant. On the other hand, Asian purchaser and investors provide new impetus.

T

he plant kingdom represents an

Roots of Technology

enormous reservoir of biologi-

Especially plant roots produce and ac-

cally active compounds with

cumulate several secondary metabolites

disease preventive, curative and

of pharmaceutical and industrial inter-

many other properties for numerous in-

est. A prime example is ginseng roots

dications. Such phytochemicals are of-

(Panax ginseng). Ginseng has emerged

ten secondary metabolites of various

from a traditional health-promoting ori-

chemical structure including alkaloids,

ental medicine drug to one of the most

steroids, flavonoids, terpenoids, tannins

popular anti-aging herbal medicines

and others. About 25 % of all pharma-

worldwide. Bioactivity is mainly as-

ceuticals on the market are based on

signed to the presence of ginsenosides

compounds derived from plants; some

(panaxosides), a group of secondary me-

are still extracted from them.

tabolites from the triterpenoid saponin

In vitro cultivation of Hairy Root Cultures on petri dish. (Picture: ROOtec)

26

Lifesciences plus 02 I 2015


SWISS RESEARCH I Phytopharmaceuticals

class with favorable effects on the cardio-

weather-independent way. “In HRCs,

hairy root and crown gall disease in such

vascular system, the central nervous

there are no such problems as plant ene-

hosts by horizontal gene transfer. They

system, as well as the immune system.

mies or plant diseases. Metabolite con-

transfer Root-inducing (Ri) extrachro-

Additional bioactive natural products

tent is always reproducible and con-

mosomal plasmids (gene-carrying struc-

like panaxynol, panaxydol and related

stant,” Harr emphasizes two of the big

tures) to the plant cells. The plasmid car-

polyacetylene compounds have shown

advantages. Hairy roots result from an

ries a part of the bacterium’s DNA

cytotoxic, antimicrobial, antifungal, an-

infection of wounded plant tissues by

(T-DNA), which is integrated into the ge-

ti-inflammatory, neuroprotective etc. ac-

Agrobacterium rhizogenes (updated sci-

nome of the host plant. Integration and

tivity and are supposed to contribute to

entific name: Rhizobium rhizogenes), a

expression of bacterial T-DNA genes

ginseng’s overall health benefit. Nowa-

Gram-negative soil bacterium. Many dif-

leads to the development of hairy roots

days, ginseng is commercially cultivated

ferent plants can serve as hosts, includ-

from the plant cells. But because this is a

mainly in plantations in northern China,

ing most dicots from the plant families

naturally occurring process, all resulting

Korea and Canada. It takes more than

Solanaceae, Rosaceae, Fabaceae, Crassu-

products – HRCs as well as extracts and

two years until the slow-growing plants

laceae,

Brassicaceae,

purified compounds – are classified as

develop flowers. Roots cannot be har-

Polygonaceae, Asteraceae and others.

non-genetically modified (nGM). It is

vested before three or four years and are

Pathogenic Agrobacterium strains cause

possible to use genetically modified

Caesalpinaceae,

best after six or seven years. Wild ginseng is particularly popular but its harvest is problematic with regard to the protection of species. Environmental pollution

and

climate

change

work

against wild and cultivated ginseng.

Hairy Root – from Disease to Technology “Chinese producers have more and more problems with heavy metals and pesticide residues in ginseng roots. This is a big problem when plants are designed for pharmaceutical use, for example in

Cultivation of Hairy Roots on large scale

Traditional Chinese Medicine”, states bi-

bioreactor systems. Roots are sensitive

ologist Jost Harr, former Vice President

to shear forces and grow best on plastic

of Research at Sandoz Agro, co-founder

or stainless-steel grids. (Picture: ROOtec)

of ROOTec AG and since 2012 ROOTec’s CEO. The many-sided businessman and experienced start-up consultant just returns from China, where he reported on

ROOtec’s CEO Jost Harr und Alicia Idoux, Head of Research, check cultivated sprouts.

ROOTec’s know-how on hairy root cul-

(Picture: Peiseler)

ture (HRC) production from ginseng. This attractive alternative to field cultivation yields secondary metabolite containing plant material in a climate- and

➜ About 25 % of all pharmaceuticals on the market are based on compounds derived from plants.

Lifesciences plus 02 I 2015

27


SWISS RESEARCH I Phytopharmaceuticals

screening

ROOTec’s bioreactor system has been developed in-house and is the centerpiece of the company’s technology platform.

A.

synergistic organisms. Receptor pro-

rhizogenes collection. After two to three

the

company’s

large

teins on the plant’s cell membranes rec-

days, cefotaxime, a cephalosporin anti-

ognize elicitors and trigger intracellular

biotic, is added in order to remove the

defense signaling, an event that often

pathogen, which otherwise would kill

goes ahead with differences in second-

the plant. Infection usually occurs in

ary metabolite production. Mechanical

one single cell, from where genetically

stress, electric pulses etc. work in the

stable, fast growing hairy roots develop

same direction. Recent reviews on hairy

after some weeks. These roots are ex-

root biotechnology, its multiple applica-

cised and then eco-friendly grown as in

tions and the current patent situation

vitro HRCs, first on agar plates in Petri

certify great potential to this new in vit-

dishes, afterwards in Erlenmeyer flasks,

ro plant biotechnology. The Swiss com-

and at last in suitable bioreactors with a

pany ROOTec, based in the Technology

capacity from 50 to 100 liters. The hor-

Center in Witterswil, about 12 km

mone-free inexpensive aqueous culture

southwest from Basel, is apparently the

medium contains mineral nutrients,

first and only company specializing in

Agrobacterium rhizogenes species in or-

sugar, and some vitamins. In some cas-

hairy root biomass production at indus-

der to produce recombinant proteins, but

es, hairy roots secrete the desired sec-

trial scale. Besides ginsenosides and po-

ROOTec is not working in this direction

ondary metabolites to the medium. Oth-

lyacetylene compounds from ginseng,

at present.

erwise, metabolites must be extracted

the six-headed ROOTec team, which

At ROOTec, HRCs are obtained from

from biomass. It is possible to increase

regularly supervises bachelor and mas-

low-germ plants, which grow from steri-

the yield of a metabolite by adding mes-

ter students from universities in Ger-

lized seeds. These plants are manually

senger substances, so-called elicitors, to

many, Switzerland and France, has de-

injured and then incubated with a suita-

the medium. Such molecules are often

veloped methods and protocols for the

ble pathogen, which is selected by

associated with plant pests, diseases or

production of antioxidants like ros-

„A small but sustainably

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“In HRCs, there are no such

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problems as plant enemies

or plant diseases. Metabolite content is always reproducible and constant.” JOST HARR. CEO at Rootec

Labgene Scientific SA – ZI Pra de Plan 35 – 1618 Châtel‐St‐Denis  021 948 02 90 – info@labgene.ch – www.labgene.ch

28

Lifesciences plus 02 I 2015


SWISS RESEARCH I Phytopharmaceuticals

marinic acid, UV protectants, antibacte-

the medium reveals starting metabolite

customer and an investor from China

rial compounds, a Vitamin D3 derivative,

production, which can be discharged

who orders products for use in Tradi-

atropine, nicotine, nor-nicotine and oth-

online, e.g. by means of a chromatogra-

tional Chinese Medicine. This is clearly

er alkaloids, cannabidiol, flavonoids, etc.

phy column bypass. Fully disposable re-

a sign that Swissness still sells,” reveals

from over 15 different medicinal plants.

actors have been developed recently. In

ROOTec’s CEO. 

Expansion is possible

order to upscale production, such bioreactors are simply set up in series. The

ROOTec’s bioreactor system has been

advantage is high flexibility and a mini-

developed in-house and is the center-

mized contamination risk. „We can easi-

piece of the company’s technology plat-

ly set up one hundred bioreactors on a

form. The so-called “Low Cost Mist Bio-

surface of 150 to 200 square meters. Ad-

reactors” consist in a stable, transparent

ditional area is available in the Technol-

60- or 100-litres single use plastic bag,

ogy Center in Witterswil“, Harr proudly

which is mounted on a steel framework

says. Anyway, further expansion re-

and sealed at the top by a reusable cov-

quires further investment. “Despite cri-

er plate. The roots, which are sensitive

sis, there is still venture capital availa-

to shear forces, are extensively growing

ble. However, investors are less ven-

on a dense grid made of stainless steel

turesome if not risk-averse, still aiming

or plastic. HRCs need humidity but do

at big profits. A small but sustainably

not tolerate stagnant water. Therefore,

growing company like ours is not con-

the medium is sprayed from a nozzle

sidered sexy enough”, notes Harr. As-

system onto the roots. The tiny fine veil

sisted by the newly established Ba-

of mist ensures optimal oxygen supply.

sel-China Business Platform, ROOTec’s

Excess medium drips off and is recy-

CEO has turned to Asian customers and

cled. Regular sampling and analysis of

investors. “I could sign a contract with a

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Lifesciences plus 02 I 2015

29

Bucher Biotec AG Viaduktstrasse 42 CH-4051 Basel Tel. 061 269 1111 www.bucher.ch


LAB & PROCESS

iStock

BIOIMAGING

Illuminating the Future Using CytoViva Hyperspectral Microscopy JAMES M. BEACH

Creating Spectral Signatures on the Nano-Scale

This was the trigger for the science of microspectrophotometery, begun amid

Making the Modern Ultra-Microspectrograph

By combining high resolution micro-

the new culture of molecular biology

The idea behind spectrographic optical

scope imaging with spectrophotometry,

emerging at the Medical Nobel Institute

microscopy has not changed, but now

it is possible to capture the optical spec-

(Reichard, 2002). Our modern drug de-

the technology behind it is vastly im-

trum of sub-micron sized materials

livery strategies now envision the use of

proved. Originally, only single spectra

within the interior of cells. This infor-

nano-sized particles that enter the cell

were obtained from point locations in

mation can in turn be used to map their

with therapeutic interventions. In 1959,

biological media, whereas in a modern

distribution and determine their mi-

Richard Feynman first commented on

imaging instrument, an entire field is

croenvironment within cell space. Tor-

the coming of this nanotechnology in

scanned to extract the spectrum at

björn Caspersson of the Karolinska In-

his APS talk titled “There’s Plenty of

every point. Using the CytoViva Hyper-

stitutet in Stockholm first developed

Room at the Bottom.” It is then not sur-

spectral Imaging (HSI) Microscope, im-

this capability for his work on cellular

prising that these two areas of technolo-

aging and spectroscopy are combined

proteins and nucleic acids, publishing “A

gy development have merged in recent

together by recording the spectrum of

Universal Ultramicrospectrograph for

times to create an important tool for de-

all points, at once, from a linear area of

the Optical Range” (Caspersson, 1950).

signing targeted cancer therapies.

the sample, and then moving the sample repeatedly by small increments to record an area as a series of lines, a process known as “pushbroom scanning.” The sample of nanoparticles, or the cells containing them, is mounted on an automated microscope stage that can be nudged under computer control in increments of 10 nanometers. A spectrographic camera is attached at the microscope view port to record spectra from the sample area “seen” through the spectrograph slit. The spectrum of each point along an imaginary sample line is thus captured while the second dimension of the hyperspectral image comes from the motion of the stage. Figure 1

Gold nanoparticles and plasmonic spectrum in live neuron. Recently hyperspectral microscopy has been aiding

shows the CytoViva Hyperspectral Im-

our understanding of cellular uptake of nanoparticles which act as carriers for the delivery of drugs.

aging Microscope system and the flow

30

Lifesciences plus 02 I 2015


LAB & PROCESS I Bioimaging

of information from images into spec-

iting plasmonic resonance (noble metal

Targeting the Cancer Cell

tral curves of imaged particles. With

NPs; Figure 3 – top left and center

Hyperspectral analysis is becoming a

megapixel CCD camera technology, im-

panels), elastic scattering (silica, titani-

tool of researchers trying to understand

age detail is recorded at the single-pixel

um oxide), coated nanoparticles (pega-

cellular uptake and trafficking of nano-

level, which at 100X magnification is

lated Au shell silica, Figure 3 – top right

particles in tumor drug delivery. Simple

equivalent to 130 nm2 of sample space.

panel), and rare earth nanoparticles

gold NP constructs (GNPs) are pro-

The spectrograph provides linear dis-

which convert low energy photons to

posed both as drug carriers and sensi-

persion of wavelengths with a resolu-

high energy photons (Figure 3 – lower

tizers to radiation therapies (Jelveh and

tion of 2.5 nm, but these wavelengths

panel). By being able to collect spectra

Chithrani, 2011). A working hypothesis

are sampled at 1.5 nm intervals on the

from single nanoparticles, particularly

is that resistance to radiation therapy

image sensor. Thus, the system actually

around their borders, hyperspectral mi-

occurs in hypoxic regions of tumors

oversamples spatial and spectral reso-

croscopy can reveal spectral changes

where ionizing radiation is not as effec-

lutions so that instrument resolutions

associated with the functionalized sur-

tive in producing oxygen free radicals

are preserved in the hyperspectral im-

face, which can be compared with spec-

that damage DNA. Thus, these tumor re-

age. After a sample is scanned by this

tra from unprocessed NPs, or after the

gions survive the therapy. If GNPs are

method, CytoViva’s software processes

drug has been absorbed. Figure 3 shows

not of themselves toxic in cells, it may

the spatial-spectral information into a

how PEG-coating diminished the scat-

be possible to augment the effects of

“data cube” allowing both spatial axes to

ter intensity in a wavelength-dependent

ionizing radiation by introducing free

appear in a color display image and

manner in Au shell silica NPs. By such

spectra to appear on the third dimen-

means, processes that include the func-

sion, which is accessible using a cursor

tionalization of the surface and the in-

[Figure 2].

tracellular delivery of drug can be moni-

HSI microscopy is now used over

tored. Past the visible range, short wave

visible and near infrared ranges to iden-

infrared (SWIR) hyperspectral micros-

tify and map biological and nonbiologi-

copy also has been used to identify and

cal materials using their absorption,

locate minute amounts of materials in-

emission and scatter spectra. These

cluding asbestos, plastics and carbon

specimens include nanoparticles exhib-

nanotubes.

02

Hyperspectral data cube arrangement. Two-dimensional color image rendered from HSI bands representing red, green and blue color channels (lower left panel). Zoom image showing a single NP (lower right panel). Spectra from NPs are on the third cubic dimension and are accessible when the cursor is positioned on the corresponding NP.

01

Diagram of CytoViva hyperspectral microscope and data flow. 1: System components include halogen light source (yellow), liquid light guide (red), enhanced dark-field illuminator (gray), motorized stage (violet), color camera (blue), spectrograph (green) and monochrome camera (tan) attached to an upright widefield microscope (white). 2: Y dimension of HSI image is caused by motion of the sample. 3: Scanned region (red rectangle) and two scan lines intersecting NPs with corresponding spectral images with wavelength spread along the vertical direction. 4: Color image of the data cube with cursors on NPs. 5: Spectra from NPs.

Lifesciences plus 02 I 2015

31


LAB & PROCESS I Bioimaging

radical promoters on GNP carriers, which are preferentially taken up in hypoxic cells. Chithrani and colleagues, who have been studying hypoxic cell uptake of GNPs, used dark-field HSI microscopy to confirm the plasmonic spectral properties of unlabeled AuNPs (see Figure 3, center panel) in the cell cytoplasm. They reported that GNP uptake in both normal and hypoxic cells caused no change in cell proliferation, but did find an increase in GNP uptake in hypoxic cells, the largest increase

03

found with 50 nm GNPs. Another area of interest is the mechanism for the accu-

Spectral plots from the CytoViva hyperspectral microscope. Plasmonic spectra from

mulation of circulating NPs in tumors.

silver and gold NP (upper left and center), spectrum (upper right) of PEG-coated Au shell

Working with living mice with intravital

on silica (white curve) compared with uncoated Au shell on silica (red and green curves),

microscopy

upconversion spectrum (lower left) of NPs excited with 980 nm laser excitation.

Smith and colleagues visualized specific

and

FACS-cell

sorting,

uptake of Cy5.5 labeled single-walled carbon nanotubes (SWCNTs) from circulating blood by a specific monocyte type (Smith et al, 2014). That the FACS-sorted monocytes contained positive SWCNTs was confirmed from spectral signatures of the nanotubes using hyperspectral microscopy, as was the finding that the nanotubes were dispersed

throughout

the

intracellular

space away from the cell membrane,

04

where they would locate if they had simply bound to the cell surface. Re-

Spectra from CCP-coated NPs in isolation (left) are mapped (right, red overlay) to the image of a cell treated with CCP-NPs. Hyperspectral image of isolated CCP-NPs in suspension (insert).

searchers have also been looking at how to introduce nano-drug carriers into cells so that they are not trapped inside endosomes as they are after endocytosis, and thus are able to more efficiently introduce drugs into cells (Pujals et al, 2009). Translocation across the cell membrane by cell surface receptors avoids the endocytotic process. Lee and Tung (2012) and others have taken advantage of this pathway by coating GNPs with arginine-rich cell penetrating peptides (CPPs), whose uptake into cells was confirmed spectrally. Figure 4

05

shows a family of spectral curves from biopolymeric CPPs in solution using

Spectroscopy and mapping of cisplatin in H&E-stained lung tissue after inhalation loading.

dark-field HSI, and the mapping of the

Family of spectra from lung tissue (left), lung tissue section showing mapped locations of drug

particles into an epithelial cell using a

(right, see text).

spectral classification technique from

32

Lifesciences plus 02â&#x20AC;&#x2030;Iâ&#x20AC;&#x2030;2015


LAB & PROCESS I Bioimaging

CytoViva called “spectral angle mapper.”

REFERENCES

CyBi®-FeliX

• Caspersson TO. (1950). A universal

Your automated application starts here

Clinical trials of cancer drugs can also benefit from spectral imaging. In a Phase II study of inhaled cisplatin for

ultramicrospectrograph for the optical range.

treatment of bone cancer that has

Experimental Cell Research 1, 595–598.

spread to the lungs, researchers are

• Reichard P. (2002). Osvald T. Avery and the

learning how lipid-encapsulated cisplatin is taken up in the affected lung tissue.

Figure

5

shows

hyperspectral

Nobel Prize in Medicine. J Biomed Chem 16(277), 13355–13362. • Neshatian M., Chung S., Yohan D., Yang C. and

curves that were collected from H&E

Chithrani D. (2014). Determining the

stained tissue that had been exposed to

size dependence of colloidal gold nanoparticle

cisplatin. These curves include the

uptake in a tumor-like interface (hypoxic).

spectra from the drug and from other

Colloids and Interface Communications. 1,

stained areas of tissue. The collection of

57–61. doi:10.1016/j.colcom.2014.07.004

curves was processed to remove spectra

• Smith BR., Ghosn EEB., Rallapalli H., Prescher

that are only found in non-treated tis-

JA., Larson T., Herzenberg LA. and Gambhir SS.

sue. The remaining curves were mapped

(2014). Selective uptake of single walled

onto areas of the treated tissue, where

carbon nanotubes by circulating monocytes for

the red overlay marks the presence of

enhanced tumour delivery. Nat Nanotechnol.

the drug.

Illuminating the Future

9(6), 481–487. doi: 10.1038/nnano.2014.62 • Pujals S., Bastús NG., Pereiro E., López-Iglesias C., Puntes VF., Kogan MJ. and Giralt E. (2009).

Hyperspectral imaging on a microscope

Shuttling gold nanoparticles into tumoral cells

is being adopted by research groups

with an amphipathic proline-rich peptide.

around the globe as a new tool to provide

Chembiochem. 10(6),1025–31. doi: 10.1002/

better understanding of targeted drug

cbic.200800843.

delivery using nanoparticles and lipos-

MipTec 2015 Stand B54

22.-24.09.2015 in Basel, Schweiz

• Sankar MU., Aigal S., Maliyekkal SM.,

omes. Other active areas of investigation

Chaudhary A., Anshup., Kumar AA., Chaudhari

include water purification (Sankar et al,

K. and Pradeep, T. (2012). Biopolymer-rein-

2012), silver toxicity, and Alzheimer’s

forced synthetic granular nanocomposites

disease (Vince and More, 2014). These,

for affordable point-of-use water purification.

and many other areas of research, will

Proc Nat Acad Sci. 110: 8459–8464,

benefit in the coming years from the

doi:10.1073/pnas.1220222110.

„

Flexible pipettor with 1 – 384 channels and automatic loading of pipetting tools

modern micro-spectrograph. CytoViva,

• More S. and Vince R. (2015). Hyperspectral

Inc. will be doing its part to ensure that

imaging signatures detect amyloidopathy

design, 12 positions on

HSI technology keeps pace with the

in Alzheimer’s mouse retina well before onset

2 levels

needs of scientific imaging work in the

of cognitive decline. ACS Chem Neurosci.

research community.  

6(2):306–315. doi:10.1021/cn500242z.

„

„

Unique and compact deck

Patent pending CHOICETM technology for pipetting from 500 nl – 1 ml

CytoViva products are represented in Europe by the Schaefer-Tec group.

„

www.schaefer-tec.com

Highest precision and accuracy for various

www.cytoviva.com

applications in all formats

AUTHOR Dr. James M. Beach. Director of Technology Development, CytoViva, Inc., Auburn, Alabama, USA

Lifesciences plus 02 I 2015

33

www.cybio-ag.com


LAB & PROCESS

METABOLOMICS

Measuring Glycolytic Function in Immune Cells Real time, kinetic measurement of glycolysis connects energy pathways to cell growth and proliferation. These results were generated by the use of an Extracellular Flux (XF) Analyzer. The technology allows simultaneous measurement of the oxygen consumption rate and the extracellular acidification rate in live, intact cells.

G

lycolysis is the cellular pro-

the

rate

science), in conjunction with the XF

cess of breaking down glucose

(ECAR) of the media, and provides

Glycolysis Stress Test, and the XF Cell

into pyruvate, providing car-

quantifiable metabolic data. This appli-

Mito Stress Test to assess the metabolic

bons biosynthesis and gener-

cation note describes the use of XF

phenotypes and function of each mu-

ating ATP. Glycolysis is utilized by virtu-

Technology to study the glycolytic en-

rine CD4+CD25- T cell subset to perform

ally all cell types in a variety of research

gine in immune cells.

glycolysis. The XF Glycolysis Stress Test

extracellular

acidification

areas. For example, dividing cells rely

Gerriets et al. (2014)2 studied mu-

was critical in illustrating the metabolic

heavily on glycolysis, and it has been

rine CD4+ T cell populations to deter-

phenotype of Teffs; whereas the XF Cell

shown to play a significant role in either

mine whether metabolic programming

Mito Stress Test was crucial in deter-

post-mitotic, mitochondrial dysfunction

affects the activation, differentiation,

mining the reliance of Tregs on oxida-

or stress. The methods of studying glyc-

and function of CD4+ T cell subsets,

tive phosphorylation.

olysis reflect the perpetually changing

specifically CD4+ effector T cells (Tef-

landscape of biotechnology: from mi-

fs), and regulatory T cells (Tregs). Teffs

XF Bioenergetic Analysis

croelectrodes, spectrophotometers, and

function in executing immunity and in-

Metabolic analyses were performed us-

NMR, to quantitative metabolic analyz-

flammation, while Tregs suppress Teffs

ing an Extracellular Flux Analyzer

ers. As the scientific research questions

to

inflammation.

(Seahorse Bioscience), which enables

that involve glycolysis have become

Therefore, a balance is crucial to main-

the real time, simultaneous rate meas-

more complex, the methods applied to

tain immunological protection and pre-

urements of oxygen consumption and

studying glycolysis have become more

vent autoimmunity. Using XF Technolo-

extracellular acidification rate (OCR

analytical and relevant to living cells.

gy, the authors were able to character-

and ECAR, respectively), by creating a

prevent

excessive

As described by the Embden-Mey-

ize the metabolic phenotypes and sub-

transient microchamber within each

erhof-Parnas pathway, glycolysis is a

strate utilization capabilities between

well of specialized cell culture mi-

series of 10 sequential reactions which

Teffs and Tregs, derived from in vitro

croplates.

break down glucose. The products of

differentiated

these reactions include protons and

cells. As illustrated in Figure 1, the CD4+

As shown in Figure 2, T cells were seed-

pyruvate, which may then be converted

T cell subsets, Teff (Th1 and Thr 17),

ed in XF Cell Culture Microplates. Cell

to lactate. The co-secretion of protons

and Treg cells have distinct metabolic

culture media was exchanged for XF

and anaerobic lactate results in the

profiles. Upon further analysis, the au-

Base Medium with no added glucose or

acidification of the media (Divakaruni

thors noted a significant reduction in

glutamine for the XF Glycolysis Stress

et al., 2014)1. In this scenario, the glyco-

the glycolytic capacity and glycolytic

Test. Glycolytic capacity was calculated

lytic pathway could be thought of as an

reserve in Tregs, in comparison to Teffs

as the difference between ECAR follow-

engine, while the glucose molecule

[Figure 1B and 1C].

ing the injection of 1 μM oligomycin,

murine

CD4+CD25- T

feeding into the pathway is the fuel. Ex-

These data indicate that Teffs will

and the basal ECAR reading. Glycolytic

tracellular Flux (XF) Technology exam-

primarily utilize aerobic glycolysis, that

reserve was calculated as the difference

ines glycolytic activity, or metabolic en-

is glycolysis in the presence of ample

in ECAR between glucose and oligomy-

gines, as well as the substrates or fuels

oxygen. In this study, the authors used

cin injections. ECAR values were nor-

utilized by the engines via measuring

an

malized to cell number.

XF24

Analyzer

34

(Seahorse

Bio-

Lifesciences plus 02 I 2015


LAB & PROCESS I Metabolomics

Summary

XF Technology allows for the simulta-

The above study shows that the differ-

neous measurement of the oxygen con-

in Switzerland at Bucher Biotec AG.

ences in metabolic phenotypes and fuel

sumption rate (OCR), and the extracel-

www.bucher.ch

utilization capabilities can influence

lular acidification rate (ECAR) in live,

www.seahorsebio.com

cell proliferation and function within

intact cells. By utilizing the XF Stress

the adaptive immune system. Studying

Test and XFp Cell Energy Phenotype

the differences, including metabolic

Test kits, researchers can obtain impor-

phenotypes and signatures, between

tant functional metabolic data and gain

these cell types may provide a viable

a greater understanding of cell metabo-

therapeutic target to maintain balance

lism. 

Seahorse Bioscience products are available

REFERENCES 1

Divakaruni et al., Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol. 2014. 547: 309–54.

or promote a desired response within

2

the immune system.

Gerriets et al., Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation. J Clin Invest. 2014. Doi: 10.1172/JCI76012

B

A 60

Glucose

Oligomycin

2DG

50

25

40

20

Glycolytic reserve

40

ECAR (mpH/min)

Th17 Treg

30 20

30 20

*

ECAR (mpH/min)

Th1

50 ECAR (mpH/min)

C Glycolytic capacity

00

25

50

75

100

125

150

*

00

00

00

10 5

10

10

15

Th1

Treg

Th17

Th1

Treg

Th17

Time (min)

01

Teffs and Tregs have distinct metabolic phenotypes (A). CD4+CD25- T cells were differentiated in vitro, passaged, and cultured with IL-2 to induce either TH1, Th17, or Tregs. ECAR was assessed following the addition of glucose, oligomycin, and 2DG (B and C). Calculations of glycolytic capacity (B), or glycolytic reserve (C).

Prior to Day Assay

Day of Assay

Isolate murine CD4+CD25- T cell Load Cartridge & Calibrate 15 minutes

y Glucose

y Oligomycin

2-DG

45 40 35

ECAR (mpH/min)

Thaw & Prepare Stock Compounds Seed differentiated T-cell subsets

Glycolytic Reserve

30 25 20

Glycolytic Capacity

15 10

Glycolysis

5 0

Non-glycol ytic Acidificatio n

0

10

20

30

40

50

60

70

80

TIME(minut es)

Run Experiment

02

Prepare Assay Medium Stock

Change to Assay Medium & Preincubate

Flow Chart of XF Assay.

Lifesciences plus 02 I 2015

35

Analyze Data

90

100


LAB & PROCESS

FOOD ANALYSIS

Serial Dilutions in Cost Effective Single-Use Sterile Bags

F

ood can become contaminated at any point during slaughtering or harvesting, processing, storage, distribution, transportation and

preparation. Lack of adequate food hygiene can lead to foodborne diseases

Microbiology testing for food and beverage safety and quality is mainly done by determining viable cell counts in homogenized and diluted samples. Instead of going through the labour intensive process of preparing test tubes for serial dilutions, users can now benefit from the sterile, single-use PE bags. Verifications by method comparison confirm the equivalence of the new method which relieves staff and increases the productivity of the testing laboratory.

and death of the consumer. Important information about the hygiene and quality of foodstuffs is the total number and types of viable cells, mainly bacteria, detected in food samples. Instead of going through the labour in-

the Zurich University of Applied Scienc-

The standard plate count method for

tensive process of preparing serial dilu-

es (ZHAW), Institute of Food and Bever-

viable cell counting is applied for more

tion tubes, laboratories can benefit from

age Innovation supported by the Swiss

than 100 years, dating back to the pio-

the many advantages of sterile, sin-

Commission for Technology and Inno-

neering microbiologist Robert Koch

gle-use polyethylene (PE) bags. The

vation (CTI).

(1843–1910). Samples to be analysed

sample for dilution is simply added into

are diluted in ten-fold steps several

the bags and sterile diluent is added by

For the verification of the dilution meth-

times with a diluent solution (e. g. ster-

just pressing a button of the Serial Di-

ods ZHAW scientists used overnight cul-

ile saline). Aliquots of the dilution steps

luter. Thereby a perfect homogeneous

tures of bacterial strains and determined

are then plated on appropriate culture

mixing of the sample with the diluent is

the count of aerobic mesophilic bacteria

medium, incubated for several hours to

achieved. The manual mixing of each

in salad, milk and minced meat.

allow colony forming and counting for

test tube with a mixer/vortexer is thus

the calculation of the number of viable

completely eliminated.

The results of replicated decimal dilu-

cells present in the original food sam-

After finishing sample dilution and

tions of the cultivated strains by the

ple. Although simple and reliable, the

plating, the used bags are removed

classical test tube method and the In-

method is very time-consuming in its

from the Serial Diluter and disposed

labtec method are shown in Figure 2

preparation

Large

[Figure 1]. Therefore, the preparation

and the results of the foodstuffs in

amounts of test tubes have to be

and examination of test tubes is no

Figure 3. The results by both methods

cleaned, filled, sealed and sterilized

longer necessary.

are equal and within the standard

and

execution.

each time before they can be used. This

measurement uncertainty of ± 0.5 log10.

consumes a lot of time, energy, water

Method Comparison

and space. The fast execution of the test

Product development and comparisons

the results obtained by using the In-

tube method also requires manual

of the Inlabtec dilution method with the

labtec Serial Diluter are equal to the re-

skills and can cause in routine physical

classical test tube method according

sults obtained by the standard test tube

discomfort or pain known as repetitive

to the international standard ISO 6887-

method.

strain injury (RSI).

1: 1992 were done in collaboration with

36

Therefore, it can be concluded that

Lifesciences plus 02 I 2015


L A B & P R O C E S S I Fo o d A n a l y s i s

Work in the labs of the ZHAW also con-

instrument is both simple and quick in

For detailed information about the

firmed that the entire dilution process is

addition to the Serial Diluters speed

Inlabtec Serial Dilution System please visit

now much less labour-intensive (no

and reliability, ease of use and its ability

www.inlabtec.com.

more laborious cleaning of test tubes)

to eliminate traditional sources of dilu-

and time-consuming. Also set up of the

tion errors.â&#x20AC;&#x201A;

02

Test results with E. coli DSM 1103, B. subtilis subsp. spizizenii DSM 347 and S. cerevisiae DSM 70449T. Shown are the mean of colony forming units of three replicates per independent series. Results of the serial dilutions with the Inlabtec Serial Diluter in blue and with the test tube method in green. Chart from the ZHAW project report.

01

Serial dilution in single-use sterile bags. A 1 ml sample together with the pipette is added into the first bag. The dosing arm is connected to the tip and the sample tenfold diluted by adding 9 ml diluent by pressing the button (a). Diluted sample is taken out for plating and for the next dilution step (b). After finishing the serial dilution

03

Total viable cell counts in samples of salad, minced meet and pasteurized milk determined by using the Inlabtec Serial Diluter (blue) and the test

and the plating of the diluted samples used bags are removed

tube method (green). Shown are the mean of colony forming units of three

for disposal (c). Watch video under www.inlabtec.com

replicates per independent series. Chart from the ZHAW project report.

Lifesciences plus 02â&#x20AC;&#x2030;Iâ&#x20AC;&#x2030;2015

37


P L AY E R S & P R O D U C T S

Advanced Monitoring and Display of Cell-Based Assays in a Microplate Reader

C

ell-based assays have always been a powerful and versatile tool in the research lab. However, these assays are quite deli-

cate as cells are very susceptible to the environment. In order to keep homeo-

Microplate readers have become multifunctional instruments that can perform measurements of complex, heterogeneous, live cell assays. Formats up to 3456-wells have allowed to significantly miniature samples to nl volumes, positively influencing throughput and costs. Microplate readers allow for all manner of reproducible, cell-based assays to be measured in seconds or minutes, depending on the density of the microplate format.

stasis, cells require specific gas and temperature conditions. Keeping cells under physiological conditions leads in fact to more robust and unbiased results, as results obtained from stressed rement data calculations can be related

particularly important in time-lapse ex-

To provide the ideal physiological

to the gas concentrations directly in

periments.

environment, microplate readers need

MARS [Figure 2]. Changes of the gas

the capability to control temperature,

concentration can be done at any point

Despite these advantages, a measu-

carbon dioxide (CO2) and oxygen (O2). In

during the experiment, either manually

rement from the top of the microplate

an effort to perfect cell-based assays in

or by software, and are shown together

without lid will always give higher sig-

a microplate reader, the German compa-

with the measurement results. Status

nal-to-blank ratios than measurements

ny BMG Labtech has developed an At-

messages like an empty gas cylinder are

from the bottom. This is mainly due to

mospheric Control Unit (ACU) for its

additionally shown in the software.

the fact that the plastic of the bottom of

cells are not trustworthy.

multimode microplate reader Clariostar [Figure

1]. The

the microplate impairs the light trans-

microprocessor-con-

In live, real-time cell-based experi-

mission, both for excitation and emis-

trolled unit can regulate CO2 and O2 in-

ments, it is beneficial to read from the

sion, resulting in lower overall signals.

dependently from 0,1 % to 20 %, making

bottom of the microplate and not from

Light reflection caused by the plastic

it unnecessary to use premixed gas cyl-

the top. Bottom reading offers several

surface and the plastic type also in-

inders to reproduce the specific physio-

advantages for cell-based detection.

creases blank values. Another factor is

logical conditions needed for cell-based

The light collector can be placed closer

the longer fiber optics required in most

assays. The integration of the ACU into

to the sample, decreasing light dissipa-

microplate readers to reach the bottom

the reader control software allows to ef-

tion. Moreover, the interfering effect of

of the microplate, since the correlation

fortlessly regulate the gas concentra-

the cell culture medium is significantly

between the length and the loss of

tions

reader

decreased. Both factors improve sensi-

transmitted light is proportional. Hence,

chamber. Moreover, the gas concentra-

within

the

microplate

tivity. In addition, bottom reading allows

much of the signal is lost when measur-

tion data are recorded over the meas-

for a cover or lid to be placed on top of

ing from the microplate bottom. This

urement time and displayed in the

the microplate to prevent cell contami-

obviously has negative implications on

MARS Data Analysis Software. Measu-

nation and liquid evaporation. This is

performance.

38

Lifesciences plus 02 I 2015


➜ Direct, Free-Air Optical Path to the Bottom of the Microplate

01

BMG Labtech has eliminated the need for fiber optics both in top and bottom

 BMG Labtech’s Atmospheric Control Unit (ACU) for all cell-based assays.

reading. BMG Labtech’s proprietary direct optic bottom reading system, found in the Clariostar LVF Monochromator reader, has set the standard in bottom reading for microplate instruments. Just like a microscope, the readers take advantage of a free-air optical path to direct and focus light onto either the bottom or top of the microplate. No fiber optics are used. This is achieved by a series of software-controlled, motor-driven mirrors. This advanced reading system displayed a significant improvement in signal-to-blank ratios when compared to readers with fiber optics, where the transit through two different mediums (air and usually quartz) is the

02

major cause of the loss of light [Figure 3]. A significant part of the light does not enter the fiber path because of re-

Gas concentration data and measurement data calculations are displayed

flection and diffraction. Further limiting

together.

factors are the width of the fiber and the amplitude angle of light collection. All these limitations are absent in free air optical paths as the mirrors allow up to 97 % light transmission. In the Clariostar, direct optic bottom reading is fully integrated into the reader’s optical system. The switch between top

and

bottom

reading

modes

is

achieved with a simple mouse click in the control software. No manual intervention by the customer, such as displacement or installation of any additional hardware, is required. All these features taken together make the Clariostar with Atmospheric Control Unit a good choice for the measurement of cellular assays. 

03

www.bmglabtech.com

Comparison of signal-to-blank ratios for top and bottom reading: GFP-tagged BAE cells were measured with a BMG Labtech reader with direct optic bottom reading and with a reader with fiber optics.

Lifesciences plus 02 I 2015

39


P L AY E R S & P R O D U C T S

HIGH THROUGHPUT SCREENING

Research for Oncological Pharmaceutical Compounds with 3-D Spheroid Cultures

H

igh Throughput Screening of

rating of the drug candidates early, dur-

Active Pharmaceutical Com-

ing the pre-clinical trial stage, is there-

pounds often still bases on

fore a considerable improvement.

conventional 2-D cell cul-

Especially in cancer research, assays

tures, even though these cultures do not

basing on spheroids are being focussed

simulate a realistic in vivo environment

on and used to replace the standard mo-

Greiner Bio-One

of a cell. This may lead to heavy losses

no-layer cultures. In spheroids, cells

microplates with

in API development. These days, 3-D

grow in a three dimensional cluster, in

cell-repellent

Spheroid Cultures have become more

which gradients of nutrients, oxygen

surface are available

and more important, since they are

and metabolites are formed, as well as

in different formats.

considered to allow better, more ration-

areas of different proliferation rates.

al and predictable in vitro experiments.

Growth conditions of an in vitro sphe-

Microplates with a cell-repulsing sur-

roid culture therefore better reflect in

face represent an ideal platform for

vivo conditions of tumour tissue4, 5, since

these 3-D cultures and they are com-

these differences in growth conditions

patible with the High Throughput Cul-

also show on a physiological level.

tivation of 3-D spheroids.

Functional differences between 2-D

Significance of Spheroid Cultures in Drug Research

and 3-D cultures regarding protein expression, phosphorylation patterns and sensitivity towards active compounds

plates with a surface effectively inhibit-

To date, the probability of a possible

have been proven to be existent in can-

ing cell adhesion are used. The latter

drug candidate being developed into an

cer cell lines.6

combine optimum surface characteris-

approved cancer drug is less than 10 %.1

tics with formats established in drug

Many API trials fail because of low ef-

Cultivation of Spheroids on Microplates

fectiveness or heavy toxic side effects

Spheroids only form if adhesion of cells

which are distributed in Switzerland by

only during the clinical trials, which is a

to the microplate surface is completely

HUBERLAB. AG exclusively, are made of

rather late phase of development.2,3 This

inhibited. Conventional cell culture sur-

polystyrol with a cell-repellent surface,

late failure represents a crucial cost and

faces, which are optimized for cells

obtained by a stable chemical modifica-

time factor within drug research. The

growing adherent or in suspension, are

tion of the starting material. 

introduction and implementation of in

therefore not suitable for spheroid cul-

vitro assays, which enable a significant

tures. For cultivation of spheroids in mi-

www.huberlab.ch

croplates either hanging-drop plates or

www.greinerbioone.com

40

screening. Greiner Bio-One microplates,

Lifesciences plus 02 I 2015


P L AY E R S & P R O D U C T S I H i g h Th r o u g h p u t S c r e e n i n g

Microscopy shows that cells adhere to conventional tissue culture surface (A), whereas they do not adhere to the surface of specially developed microplates with cell-repellent surfaces (B).

REFERENCES 1

Hutchinson L, Kirk R (2011) High drug attrition rates – where are we going wrong? Mat Rev Clin Oncol 8:189–190.

2

Hopkins AL (2008) Network pharmacology: the net paradigm in drug discovery. Nat Chem Bio 4:682-690.

3

Kola I (2008) The state of innovation in drug development. Clin Pharmacol Ther 83:227–230.

4

Friedrich J, Seidel C, Ebner R et al. (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324.

5

Kunz-Schughart LA, Freyer JP, Hofstaedter F et al (2004) The use of 3-D cultures for high-throughput screening: the multicellular spheroid model, J Blomol Screen 9:273–285.

6

Eker JE, Johnson K, Strake B et al. (2014) Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro – implication for drug development PLoS One 9:e92248.

Lifesciences plus 02 I 2015

41


P L AY E R S & P R O D U C T S

BIOPROCESSING

Tailored Cell Culture Media – Produced in a Cleanroom of GMP Standard Production of sterile high quality media of big batch sizes requires a lot of technology. BioConcept, manufacturer of the Amimed tissue culture media, invested in a new cleanroom of pharmaceutical standard and a water purification system in order to produce Water for Injection, liquid and powder cell culture media of the highest quality available for customers. A GMP certification is in process.

W

hen working in a biological

Liquid II goes GMP Cleanroom

sary as well as exact implementation

laboratory with cell cul-

August 2015 represents a further mile-

and validating.

tures, one gets in touch

stone in BioConcept’s history: In the

The new plant was designed to cre-

with cell culture media,

course of development and progress,

ate a surrounding that ensures a high

powdery ones, to prepare with water, or

the enterprise designed and built a new

degree of sterility, ensuring a sterile fi-

liquid ones, ready to use. Manufacturer

production line called Liquid II, which

nal product. A state of the art air pro-

of such media in Switzerland is BioCon-

is a liquid media plant. Liquid II is cur-

cessing system is used to supply the

cept. The company was founded in 1978

rently approaching its completion and

optimal conditions needed for sterile

and has therefore almost 40 years of ex-

will be inaugurated. The new automated

liquid production. The air-conditioning

perience in serving the Swiss biological

liquid handling processing line meets

is a very important factor in the pro-

community with labware – since 1993

pharmaceutical GMP standards. It is

cess of manufacturing pharmaceutical

also with an own brand of tissue culture

made of stainless steel, is situated in a

substances. Pressure, temperature, hu-

products and media, the Amimed prod-

GMP class A (or ISO class 5) cleanroom

midity and particle count in the air are

uct line. BioConcept has become a lead-

and has a batch capacity of up to 5000 l/

constantly monitored and controlled in

ing supplier and service partner for nu-

day. There are many requirements to

order to maintain air quality of GMP

merous reputable pharmaceutical and

meet,

cleanroom.

class A  /  ISO class 5 standards. The

academic institutions in Switzerland.

Therefore, intensive planning is neces-

monitoring improves consistency of the

when

building

a

The purpose with Amimed was to manufacture tissue culture products for the sophisticated and evolving pharma-

A broad range of special and standard media are

ceutical and bio-pharmaceutical markets. With its product range, BioConcept has developed a strong international presence. Nevertheless, the company is still situated in Basel in Switzerland. In

manufactured in order to create the ideal cell

growth medium and other sterile liquids tailored

Allschwil, a broad range of special and

to meet the customer’s needs.

standard media are manufactured in order to create the ideal cell growth medium and other sterile liquids tailored to meet the customer’s needs.

42

Lifesciences plus 02 I 2015


P L AY E R S & P R O D U C T S I B i o p r o c e s s i n g

procedures or detects early possible deviations that may arise. A regular H2O2 disinfection ensures sterility of

01 02

the cleanrooms. The Hydrogen Peroxide Vapour (HPV) equipment basing on spinning disk technol-

Glimpse at the drying room and its airlock. The cleanroom

ogy is used to produce a fine fog of uniform and controlled droplets of 5–10 µm size. The machine disinfects the designated space effectively without

The automatic filling line can fill up to 2000 500 ml bottles

using nozzles or compressed air. The rooms and facility have been integrated in

03 04

the original building. 1000 m2 of new production and storage space offers the operators to work in

of media in one hour. For the preparation of the bottles a cleanroom robot is used, which assures a high standard

Detail of filling line. The motors and moving parts of the

an environment equipped with the latest technologies and materials. The working place is spacious and easy to access, thus the plant can be run con-

automatic filling machine are oil free.

The filling machine is located in the centre of the plant and

veniently and the products are rapidly transferred

Lifesciences plus 02 I 2015

is designed to enable a smooth workflow of product and personnel.

made almost entirely of stainless steel.

01

02

03

04

43


P L AY E R S & P R O D U C T S I B i o p r o c e s s i n g

to the appropriate storage due to the close proximity of the new storage rooms. A large proportion of the interior has been made from well-sized windows. A lot of glass has been used in order to make the plant more open and transparent. This layout is

Interview

arranged to make it possible to inspect one side of the plant whilst standing on the other. The transparency concept allows the staff to easily overlook

5 Questions from Life Sciences plus addressed to Martin Howald, CEO of BioConcept

the whole production process, which in turn is monitored by state of the art sensors and visualised on several user interfaces, integrated into the plant’s structures. The company’s offices are also in close vicinity, on the floor above. With the location, the interior and automation of the processes, BioConcept has

LS+ (Sonja Bichsel): Mr. Howald, what was the motivation

increased managing and monitoring of the manu-

for BioConcept to invest a lot of money and build the high

facturing process, in order to produce quick but

standard production line Liquid II?

with highest quality possible. For custom made products, delivery time is

Martin Howald: We had to react to the increase in inquiries we were receiving from the cell culture market. Our existing

about six weeks upon order. These products can in-

production plant, Liquid I, only has manual filling, so we

clude customer designed media (powder or liquid),

were unable to fulfil all inquiries. So the logical consequence

individual solutions for cell cultures, cell system

was to increase and automate our production capabilities.

applications, buffers and balanced salt solutions or supplements and auxiliary reagents. The products

LS+: Who are the typical customers ordering BioConcept’s media?

are then manufactured in accordance to the cus-

Martin Howald: Our typical customers work in research as well

tomer’s recipe. Sterilisation can be done by filtra-

as biopharmaceutical and pharmaceutical production worldwide.

tion with 2 µm filters or by vapour sterilization.

Customers of products produced by Liquid II will not differ from

Customers can also choose the container for the

our current customer base, but as we are specialized in customized

product filling: a variety of size and types including

products, we are open for all other inquiries beside the usual

glass, sterile bags or PET bottles of different volumes. Batch sizes range from 5 to 5000 l or 2 to

customer applications.

800 kg, respectively. LS+: A project like Liquid II is time consuming, how long did it take to realize it?

Water and Vapour

Martin Howald: It took us one year from the start of planning

Chemically and microbiologically pure water is a

to the final completion. All the planning was executed in-house,

key component in manufacturing liquid biophar-

we did not work with external architects or engineering companies.

maceutical medium. Therefore, BioConcept invested into a new water purification facility. The system

LS+: What are the major challenges when building a new

is engineered to efficiently generate Water for In-

production line?

jection (WFI), water of the highest approved phar-

Martin Howald: The major challenge was the short time

macopoeia standard. The facility now produces up

frame of 1 year and – concerning the given building structure –

to 5000 l of media per day, which meets the rising

the implementation of Liquid II without disturbing our running

demands of the customers. The process to transform regular tap water to

production.

WFI usually starts with reverse osmosis. Quality of LS+: What significance does a GMP certification have for the

the resulting highly purified water has to be moni-

biotechnological and life science industry?

tored constantly. Therefore, pH and osmolarity as

Martin Howald: GMP plays a key role for our customers working

well as conductivity, endotoxin and bioburden lev-

in biopharma and pharmaceutical production. As a supplier for these

els have to be determined and recorded. The purified water is either integrated in the

customers we need to have a GMP certificate in order to be accepted as a trustworthy supplier. We started already in 2005 in our ISO 9001

cleaning system of the facility or is turned into pure

certificate with working according to GMP standards. For the new

steam by a powerful electric steam generator. For

Liquid II plant we will achieve a GMP certificate.

production of WFI, the pure steam is condensed. During this process the heat is extracted and recycled into the air conditioning system. It is then used

44

Lifesciences plus 02 I 2015


P L AY E R S & P R O D U C T S I B i o p r o c e s s i n g

With its product range, BioConcept has developed a strong international presence.

The three new media filling stations can handle up to three 500 liter containers simultaneously.

to control the humidity and temper-

tainers. The modern sensors and

ature of the rooms. WFI has to be

equipment are used to assure a high

kept at a constant temperature

level of accuracy and flexibility

above 80 °C to avoid microbiological

when measuring product parame-

contamination. To maintain a sus-

ters. Its capacity ranges from 100 l/

tainable and constant temperature,

day to 5000 l/day and has the possi-

the energy from the heat exchang-

bility to handle various bottle and

ers is recycled around the water cir-

container types from 100 ml to 500 l.

cuits. This is monitored constantly

The automatic filling line can fill

using sensors. Besides WFI produc-

up to 2000 500 ml bottles of media in

tion, the generated steam has other

one hour. For the preparation of the

purposes within the manufacturing

bottles, a cleanroom robot is used,

process: it is connected to the

which assures a high standard. All

Clean-in-Place / Sterilize-in-Place

further steps, such as sealing and

(CIP/SIP) systems and the sterilizer.

labelling, are also performed auto-

The use of an electric steam

matically. The motors and moving

generator instead of the commonly

parts of the automatic filling ma-

used petrol powered alternative re-

chine are oil free, which reduces the

duces carbon emissions of the com-

risk of contamination.

pany. The loops of purified water

The three new media filling sta-

and steam for cleaning and heating

tions can handle up to three 500 lit-

purposes save electricity and there-

er containers simultaneously. This

fore contribute to the plant being

allows completing even big batch

more ecological and financially sus-

sizes within one day. The media

tainable.

pipes and tanks are made out of

Automated Production A high quality vapour sterilizer is

pharmaceutical standard electropolished stainless steel and are linked to the CIP/SIP system.

designed to disinfect solid and porous products such as filters, rub-

BioConcept is proud of their new

ber stoppers, and system compo-

state of the art liquid media plant

nents. The sterile filters are cus-

called Liquid II. The plant has

tomarily disinfected in order to

been designed to maximize efficien-

avoid cross-contamination. Integri-

cy, sustainability and productivity

ty tests monitor each of the filters

through utilizing modern technolo-

to make sure they are operating

gy and focusing on the fine detail of

correctly. This represents a reliable

the plants design. The design of the

GMP-compliant design.

new plant and the equipment used

In their new facility, BioConcept

are currently in accordance with

uses state of the art machinery such

GMP guidelines and the company is

as an automatic filling system as

in the process of being certified. 

Nano-Glo ® Dual-Luciferase Reporter Assay ®

See what you have been missing with the Nano-Glo® Dual-Luciferase Assay–A reporter partnership sensitive enough to detect endogenous expression, bright enough to let you see things as they really are, and flexible enough to let you make all the choices.

www.promega.com/go_nano It’s the little things that make all the difference.

well as three media filling stations that are designed to fill large con-

Lifesciences plus 02 I 2015

www.bioconcept.ch

45 © 2014 Promega Corporation. All Rights Reserved. 15108087


P L AY E R S & P R O D U C T S

CELL CULTURE

Paving the Way for Cell Fate Discovery

Currently, there are two approaches to differentiating cells that hold great promise in the field of regenerative medicine. In one approach (iPS), somatic cells (such as fibroblast cells from skin) are reprogrammed into pluripotent stem cells that have the potential to differentiate into any cell type in the body (such as neurons, heart, pancreatic, and liver cells). Another approach

R

(transdifferentiation) reprograms maeproducible cell culture is a

quently results in long laborious days at

ture somatic cells directly into other so-

cornerstone of basic research,

the bench for researchers, a need to lim-

matic cell types without reverting to a

drug discovery, and cell thera-

it the number of chemical and physical

stem cell stage. Although both ap-

peutics

the

steps, high-heterogeneity, and contami-

proaches have great scientific and ther-

emerging fields of regenerative medi-

nation that can make results difficult to

apeutic potential, many of the existing

cine and stem cell research, scientists

interpret. Safe, simple, precise and re-

protocols are difficult to reproduce and

rely heavily on in vitro culture to mimic

producible protocols are key to the suc-

result in highly heterogeneous sample

in vivo cellular environments to develop

cess of realizing the vast potential of

populations.

more efficient and predictive disease

stem cell and cell-based therapy.

discovery.

In

models. However, to date, much of this work

To solve this growing problem, Fluidigm, with support from the California

is done using manual procedures that

Institute

fail to achieve consistent results due to

(CIRM), developed a microfluidic cell

complex, multi-stage protocols. In stem

culture chip and support system to ac-

cell fate studies, scientists are often left

celerate stem cell research, with the

with frequent pipetting and plate han-

hope that a better understanding of

dling steps, involving dozens of chemi-

what leads stem cells to differentiate in-

cal and physical steps that must be per-

to desired types of cells would be useful

formed in a precise protocol over a peri-

in the development of therapeutic ap-

od of several weeks. This complexity fre-

plications.

for

Regenerative

Medicine

➜ “Creating the optimal conditions for reprogramming and differentiation, stem cell culture and stem cells has

Callisto IFC

historically been a tedious and manually laborious task. This system allows a user to more efficiently test a variety of cellular stimuli at various times without having to stay tied to the bench.” LILA COLLINS. PhD, CIRM Science Officer

46

Lifesciences plus 02 I 2015


P L AY E R S & P R O D U C T S I C e l l C u l t u r e

hiPSCs cultured in the Callisto IFC can be automatically fed, dosed, stained, and harvested with minimal handson time. A) hIPSCs are transfected with mRNAs and Cy3-labeled siRNAs, B) nGFP mRNA, siRNA against nGFP, siRNA negative control, C) Immunostaining with OCT4 antibody revealed significant decrease in OCT4positive cells after knockdown with anti-OCT4 siRNA.

The Callisto system will allow research-

flexible treatment and readout of indi-

or run at separate facilities, reducing

ers to develop simpler and safer proto-

vidual chambers at user-defined time-

operator-to-operator variability.

cols with more easily controlled out-

points throughout the experiment.

comes. Callisto addresses these issues

With the expanded capabilities offered

directly with hands-free multifactorial

Callisto streamlines experiment design

by the Callisto system, researchers can

dosing and manipulation in 32 inde-

and execution through an intuitive

accelerate our understanding of com-

pendent cell culture chambers. Each

touchscreen interface and accompany-

plex biological processes that drive cell

chamber can culture dozens to thou-

ing experiment planner software. The

development, disease onset and pro-

sands of cells, and can be dynamically

workflow enables a user to schedule any

gression by focusing on experimental

dosed with up to 16 different factors for

type of culture event – such as media

design and data analysis rather than

as long as three weeks. The system sup-

feeding, combinatorial dosing, tempera-

dealing with the inconvenience and

ports integrated protocols to culture,

ture or gas changes, staining, lysis and

limitations of basic cell maintenance

dose, stain, harvest and lyse cells within

harvesting – during the course of the

and optimization. 

each chamber, and provides dynamic

cell culture protocol, for up to three

dosing and harvesting of some or all of

weeks. The resulting scripts can be

www.witec.ch

the chambers simultaneously to enable

shared with colleagues for optimization

www.fluidigm.com

Lausanne 2016

Expo Beaulieu

Lausanne 13 & 14 April 2016

The show for laboratory technologies & services Lifesciences plus 02 I 2015

47


P L AY E R S & P R O D U C T S

Uncover and Handle Details at the Nanometer Scale

T

he Schaefer Group distributes

sive. The new “NanoAssemblr “platform

image of the sample can be captured in

over Continental Europe equip-

by Precision NanoSystems Inc. is a mi-

minutes, providing the spectral re-

ment for basic as well as applied

crofluidics-based system for the devel-

sponse within each nanoscale pixel of a

research, the industry and edu-

opment and manufacture of liposomes

high resolution image. By comparing

cation, in the field of micro- to na-

and nanoparticles for drug delivery ap-

the spectra with a spectral library, the

no-technologies and surface and engi-

plications. The NanoAssemblr uses au-

particles and molecules are chemically

neering and metrology. The following

tomated instrumentation to formulate

identified.

examples show the extensive range of

nanoparticles by nanoprecipitation us-

products and experience.

ing millisecond mixing of nanoliter re-

05 Single Cell Manipulation

action volumes. This well-controlled

“FluidFM” is a newly developed and

process mediates self-assembly of nano-

patented technology by Cytosurge AG

Dynamic Light Scattering (DLS) allows

particles with reproducible sizes and

that

particle sizing down to 1 nm diameter.

low polydispersity. At the same time it is

nano-manipulator via a hollow cantile-

Typical applications are emulsions, mi-

easily scaled by parallelizing microflu-

ver with micro-fluidics. The cantilevers

celles, polymers, proteins, nanoparticles

idic mixers, which maintains identical

with a tiny opening at the tip can be

or colloids. A major drawback of con-

reaction conditions for batch sizes from

used for various single cell manipula-

ventional DLS is its restriction to trans-

millilitres to litres. Individual microflu-

tions: injection of liquids into the cell,

parent samples since multiple scatter-

idic mixers are capable of producing

extraction of liquids from the cell,

ing in turbid or opaque samples can

LNP at 1—24 ml/min. Implementing a

measurement of adhesion force with

lead to measurement errors by orders of

continuous flow pumping system allows

pico-Newton

magnitude. The new, patented, 3-D

for siRNA-LNP batch sizes in the 25—

and electrophysiology. Due to its auto-

cross-correlation technology built into

500 ml range for pre-clinical studies.

mation capabilities and its ease of use,

01/02 DLS and Microrheology

the “NanoLab 3D” instrument by LS In-

combines

a

force

resolution,

controlled

pick & place

the technology will revolutionize single

struments AG is a powerful technique to

04 Label-Free Nanoscale Imaging

suppress multiple scattering and thus

The “Hyperspectral Imaging system” by

a crucial role in areas like proteomics,

measure concentrated samples reliably.

CytoViva, Inc. is based upon a patented

metabolomics, drug discovery, cell me-

Sample dilution is no longer required!

Enhanced Darkfield Optical Microscope

chanics and stem cell research.

Hyperspectral

The FluidFM technology can also be

monitors the thermal motion within a

Analysis (VNIR or SWIR range). It pro-

used as a lithography tool and with an

sample with nanometer resolution using

vides label free bio-molecule, exosome

integrated electrode, to patch clamp

Diffusing Wave Spectroscopy (DWS) and

and nanoparticle determination in bio-

moving and delicate cells.

automatically translates this information

logical matrixes.

Optical

rheology

(microrheology)

with

high

resolution

cell experiments and is expected to play

into rheological data. The DWS “Rheo-

The CytoViva Hyperspectral Micro-

For the discovery of details at a nano-

Lab tabletop” instrument by LS Instru-

scope System allows researchers to

meter scale, Schaefer-Tec AG distrib-

ments AG is the fast and easy-to-use La-

spectrally confirm and spatially deter-

utes devices and technology of

ser Light Rheometer based on DWS.

mine the location of drugs and nano-

03 Nanoparticle Production

particles in cells without the use of a fluorophore.

Traditional lab-methods for producing

Within seconds, one can optically

lipid nanoparticles (LNP) are inconsist-

observe nano-material samples in al-

ent, difficult to scale, and labor-inten-

most any environment. A hyperspectral

01

02

03

04 48

• www.lsinstruments.ch • www.nanoassemblr.com • www.cytoviva.com • www.cytosurge.ch www.schaefer-tec.com

05 Lifesciences plus 02 I 2015


P L AY E R S & P R O D U C T S

A revolution in cryogenic sample storage “Today’s vial products are limiting the outcome for success”

C

Usual fixed-volume cryotubes have major drawbacks:

ryogenic storage of biological

• Increase: Space / Contamination / Mix-ups / Resources

material is a standard procedure

• Decrease: Stability / Quality / Biomarker Detectability

in many labs. Plasma, urine or other samples need to be frozen

and kept for long time, to be available for analysis sometime in the future. For this purpose, special vials are used that can stand temperatures down to –196 °C. The

standard procedure for cryogenic storage of a sample is using a large vial that is frozen and thawed multiple times, or aliquoting it into several smaller vials, freezing them and thawing one vial at a time. However, these procedures have obvious disadvantages. Multiple freeze and thaw cycles of large vials lead to decrease in quality and stability of the

FlexiQuot tubes with its breaking procedure without sample contact or thawing leads to better storage and sample stability.

samples. Dividing the sample into smaller vials is time consuming and prone to

FlexiQuot combines the flexibility of

snapping tool for FlexiQuot tubes allow

human errors in labeling and storage,

multiple small cryogenic vials with the

the breaking procedure without contact

and also leads to sub-optimal use of pre-

storage and cost effectiveness of one

to the sample surface. Thus, the highest

cious cryogenic storage space.

large cryotube. It is the first dividable

quality of the sample over time is en-

plastic cryotube in the world, with a to-

sured and contamination is avoided. 

tal volume of 5 ml that can be separated

Flexible cryogenic storage

into 5 aliquots of 1 ml. The dividable

“The solution is using dividable vials”

cryotube helps you optimize frozen sample storage. It gives its user the ability to hold multiple samples in one vial

Therefore, as experienced researchers

and several caps that can cover both

and

claim,

sides of the snapped aliquot and the re-

there was definitely a need for a better

maining sample. This way, you can

cryotube. FlexiQuot was developed to

break off the aliquot that you need, cap

address the need for a flexible and

it and put the rest back in the freezer.

more efficient cryogenic storage tube

The user does not have to thaw the con-

that maintains the maximum quality of

tent of the entire vial for an analysis.

a frozen sample in long term.

Furthermore, the special holding and

laboratory

professionals

Each aliquot has bayonet pins in the upper and lower part and this is part of the sealing mechanism. In the lower part the bayonet is designed in a way that when sealed, the lid is locked and cannot be unlocked again, to avoid any spilling of the sample. There is also an arrow that indicates the upper part of the aliquot, namely the lid to open. In the first aliquot there is also a line indicating a total volume of 5 ml above that the user should not fill. The user can identify the breaking points on the tube, where the plastic is thinner, to facilitate breaking.

Lifesciences plus 02 I 2015

49

www.1cryobio.com


P L AY E R S & P R O D U C T S

TOOLS First All-in-One Spectrophotometer / Fluorometer DeNovix Inc., a developer and manufacturer of life science instrumentation for bio-research launches the DS-11 FX+ Spectrophotometer/ Fluorometer. The device combines patent pending technologies to deliver the most flexible and complete system available for nucleic acid and protein quantification. DeNovix SmartPath Technology allows rapid and accurate 1μL UV-Vis quantification over an industry-leading dynamic range. The fluorescence mode now also enables researchers to measure picogram amounts of sample with increased specificity in one compact instrument. The third operation mode utilizes quartz or plastic cuvettes to perform full UV-Vis measurements for kinetics and cell quantification. The DeNovix platform provides researchers a wide range of commercially available or custom assays. The DS-11 FX Series includes DeNovix’s custom Android operating system and a high-resolution, glove-compatible touch screen. The intuitive EasyApps software provides built-in standard parameters for the most common absorbance, fluorescence and colorimetric assays. DeNovix EasyApps for quantification of DNA, ssDNA, RNA, purified proteins, labelled proteins, and other spectrophotometric and fluorometric measurements come pre-installed. DS-11 FX+ includes Wi-Fi, USB, and Ethernet connectivity, allowing users to easily export data via e-mail, a USB drive, to network drives or printers, or print cryotube labels with an optional printer. Bucher Biotec AG | www.bucher.com

AL4: Customised, high precision, digital low pressure sensor

AL4: Customised, high precision, digital low pressure sensors for medical applications Pewatron introduces the new Fujikura digital AL4 series of gauge low pressure sensors for volume production. The AL series has a very small footprint (11.36 mm × 10.32 mm) in comparison with other low sensors, SMT pin configuration and a high total accuracy (+/- 1.5 % FSO) within a compensated temperature range of 0 °C to 50 °C. The digital AL4 series is a two-chip system consisting of a piezo-resistive pressure sensing chip with a very high maximum load pressure of 100 kPa and an ASIC signal conditioning chip. The ASIC allows digital signal conditioning and communication via the I2C protocol. The digital AL4 series low pressure sensor product follows the successful introduction of the analogue, high precision A2 series pressure sensor and the digital, high precision A4 series pressure sensor. The heart of the ASIC is a 14-bit ADC, which achieves the equivalent sensitivity of up to 0.15 Pa/LSB for a 2 kPa full-scale sensor. Six slave address codes can be assigned to the pressure sensor for individual communication with the microcontroller master. The sensors are delivered in the usual high quality Fujikura packages comprising tray, tape and reel, according to customer specification. Standard measurement ranges are between 0 kPa to 2 kPa and 0 kPa to 10 kPa; non-standard ranges are available on request. The pressure range is configurable as positive, negative or bidirectional gauge. Supply voltage is configurable at 3.0, 3.3 or 5.0 VDC. Pewatron AG | www.pewatron.com

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P L AY E R S & P R O D U C T S I To o l s

Cell Energy Phenotype Test Determines Metabolic Potential Seahorse Bioscience introduces the XFp Cell Energy Phenotype Test Kit. The kit enables a unique real-time assay on live cells that determines their baseline metabolic phenotype and potential. This one-hour test measures both, the mitochondrial and glycolytic activity of the cells, and compares their baseline values with metabolic activity under stressed conditions, induced by a single injection, to determine the metabolic potential – the cells’ ability to respond to an energy demand. This kit has been designed specifically for use with the XFp Extracellular Flux Analyzer. Traditionally, mitochondrial respiration and glycolytic activity is obtained from a number of separate assays, with the comparison performed after the assay completion. The new XFp Cell Energy Phenotype Test is the only method available that can provide a metabolic phenotype with which scientists can make direct, functional comparisons of both metabolic pathways between groups of live cells. By simultaneously measuring the relative utilization of the two major energy pathways under both basal and stressed conditions, researchers can now quickly realize the metabolic consequence of genetic changes. With a better understanding of the connection of physiological traits of cells with genomic and proteomic data, scientists are generating new insights into metabolic function, leading to a greater understanding, and new treatments of disease. There are now over 1,500 references to the Seahorse XF Technology in published articles in leading scientific journals. Bucher Biotec AG | www.bucher.com

LIFE SCIENCE Brenntag Schweizerhall Inc. Business Unit Life Science Elsässerstrasse 231, CH-4013 Basel Tel.: +41 (0) 58 344 80 00 Fax: +41 (0) 58 344 82 08 www.brenntag.ch Business Manager Life Science Beatrice Del Principe Tel.: +41 (0) 58 344 86 68 beatrice.delprincipe@brenntag.ch Lifesciences plus 02 I 2015 www.brenntag.ch

From producing food that serves as a vital source of nutrition and energy to manufacturing pharmaceuticals that cure illnesses and keep us in good health, companies in the life science industry manufacture products and provide services that are of fundamental significance to modern society. Brenntag fully understands the importance of making sure that these offerings are undertaken with the highest quality and most effective products on the market. Over the years, Brenntag Life Science has emerged as the specialty chemical industry’s number one partner when it comes to the distribution of products and services that have an indispensable impact on a company’s performance in the life science industry. Life Science at Brenntag

encompasses a variety of business units that offer our customer high-quality ingredients, tailor-made supply chain solutions and an unrivaled degree of technical and market expertise. Partnering with Brenntag Life Science, one of three divisions at Brenntag along with Material Science and Environmental, means that your company is aligned with a true industry leader, one with a presence in every European market who understands the importance of always staying one step ahead of the curve. Our division at a glance Brenntag is the distributor of choice for companies and suppliers in the specialty chemicals industry. The Life Science division at Brenntag incorporates our 51 renowned Food & Nutrition, Animal

Nutrition, Cosmetics as well as Pharma business units. Brenntag Life Science is made up of an experienced team of sales representatives and technicians who consistently rank amongst the best in their field for expertise and professional service. Brenntag has a distinguished presence in every European market, with over 210 distribution centers in more than 30 countries and an extensive network of specialized application centers across Europe. We aim to create value for every one of our customers and suppliers, irrespective of the application or business challenge at hand. Brenntag Life Science is committed to fulfilling all of your requirements by offering you the widest range of products, most innovative and sustainable solutions and best technical expertise on the market.


Cutting Edge Innovation in Applied Life Science Research The life science research community is at a highpoint and rising â&#x20AC;&#x201C; where real-world applications of breakthrough fundamental research are proceeding at rapid speed. The next Disruptive Innovation Revolution is taking place where these innovations are applied to improve human health. For example, the billions of dollars in investments in cell based treatments are providing astonishing treatments in leukemia using precision immunotherapy. Crispr based gene editing technologies are ushering in the revolution of precision genomics to correct disease-causing genes while lab protocols for rapid whole genome sequencing are being employed in hospitals for precision medicine. A tremendous opportunity exists for research scientists in all fields to drive this revolution and maximize the gains. Since innovation happens at the crossroads of disciplines, the Basel Life Science Week (BLSW) provides an environment in the heart of Europe to foster interaction between scientists from academia, industry and non-profit groups. In the city by the Rhein river, participants exchange with world class speakers and explore new avenues for applications across multiple fields of research. The Basel Life Science Week conference will be held from September 21 to 24, 2015, in the Congress Center of Basel, Switzerland. The keynote lecture series hosts notables such as the CEO of Actelion, Dr. Jean-Paul Clozel, Founder of Breakout Labs and former CEO of PayPal, Mr. Peter Thiel, Head of R&D at Roche, Dr. John Reed, and former VP of Biomarkers at Bayer and Professor at Charite, Dr. Kushru Asadullah.

The 13 science Forums host over 150 international speakers. Forum topics for 2015 include Digital Health and Care, Aging and Drug Discovery, Translational Medicine, Medicinal Chemistry, Stem cells, Synergy in Drug Combinations, Infectious Diseases, Peptide Therapeutics, Signaling and Drug Resistance in Cancer, and many more. Every year, the program committee receives over 150 high quality science abstract submissions for poster presentations. Many are selected as talks in the various Forums. To highlight innovative submissions, the committee rated and selected 10 abstracts for oral presentation in a unique Forum setting. Our community services program brings in speakers for career counseling for jobs in the life sciences fields. This year, we host Peggy McKee, a renounced high powered speaker from Career Confidential. She will present a full day workshop on how to differentiate you in your job search and network for hidden jobs. Combined with the 10 Industry sponsored Forums, and the 100+ Vendors in the exhibition hall, the Basel Life Science Week is truly a showcase of innovation across multiple disciplines. We hope to see you at this yearâ&#x20AC;&#x2122;s exciting event. If you have any questions or would like to reach the Basel Life Science Week secretariat, please visit the Basel Life Science Week website (www.basel-life-science-week.eu) or write to registration.blsw-miptec@congrex-switzerland.com. Dr. Bhupinder Bhullar | Chair of Steering Committee Basel Life Science Week 2015

Dr. Bhupinder Bhullar Chair of Steering Committee Basel Life Science Week 2015


Tools at MipTec Cytation 5 Cell Imaging Multi-Mode Reader BioTek’s Cytation 5 Cell Imaging Multi-Mode Reader is a uniquely integrated, configurable system that combines automated digital widefield microscopy with conventional multi-mode microplate detection to provide phenotypic cellular information and well-based quantitative data. This single instrument platform replaces multiple modules and software interfaces, yet is simple to set up and operate. With up to 60x magnification, the microscopy module provides highquality cellular and sub-cellular visualization in fluorescence, brightfield, color brightfield and phase contrast channels. Available laser autofocus and image-based autofocus ensures fast and accurate image acquisition with minimized phototoxicity across a broad application range.

Future-Proof Liquid Handling Automation CyBi-FeliX is a liquid handling platform with 1 – 384 channels in the volume range from 0.5 to 1000 µl. The high-precision parallel transfer in 96-well or 384-well format is complemented by pipetting in single wells, as well as the pipetting into columns and rows. CyBi-FeliX offers maximum flexibility with minimal space requirements through a unique deck design with 12 positions on 2 levels. Thus, CyBi-FeliX provides sufficient space for microplates, tubes as well as shaker, magnet adapter and gripper. The modular concept of CyBi-FeliX enables customized configurations for a wide variety of applications and can be adapted at any time to changing requirements.

The multi-mode detection module features BioTek’s patented Hybrid Technology, which incorporates variable bandwidth monochromator optics and high sensitivity filter-based detection optics for unmatched versatility and performance. Temperature control to 65 °C and shaking, plus available CO2 / O2 control and dual reagent injectors optimize conditions for cell-based imaging and detection. Image capture, data collection and powerful image and data analysis are managed with Gen5 software, specifically designed for uncomplicated processing of even the most complex assays.

The successful automation of Cisbio HTRF assay is an example of the active ingredient search for the complex analysis of cellular signalling pathways. The method includes serial dilutions of test substances, parallel transfers of reagents and addition of substrate solutions in rows and shows the flexibility of CyBi-FeliX.

 ioTek Instruments GmbH Switzerland | B www.biotek.com

Visit us at MipTec Booth D36

Analytik Jena AG | www.analytik-jena.com

Visit us at MipTec Booth B54

Lifesciences plus 02 I 2015

53


Simplifying Measurement of Absolute Quantum Yield To improve OLEDs, LEDs, solar cells, bio-labels, etc. it is key to know the photoluminescence absolute Quantum Yield. This figure gives the ratio of emitted and absorbed photons. It tells how effective a dye converts excitation light into fluorescence light. Lab measurements so far took hours and required reference standards. The Hamamatsu Quantaurus QY is a game changer enabling measurements of absolute Quantum Yield reproducibly within minutes for liquid solutions, powders and thin film samples – without reference samples. Dye samples are simply placed inside an integrating sphere, then single excitation wavelengths are measured or scans over selectable spectral ranges are performed. The instrument quantifies absorption at each excitation wavelength, delivers fluorescence spectra and absolute Quantum Yield values on the fly.

Accelerate Suspension-Cell Screening and Combinatorial Profiling for Immunological Applications IntelliCyt Corporation launched the iQue Screener PLUS. The iQue Screener platform is an integrated instrument, software, and reagent system that enhances the screening workflow from sample preparation through results. It enables rapid, high content, multiplexed analysis of cells and beads in suspension in 96, 384, and 1536 well plates. Due to its patented sample delivery system, plates are processed rapidly (less than 5 minutes for 96 wells) and assays can be miniaturized to conserve precious sample and use less reagent per well. Software-assisted automation, analysis, and experiment-level visualization tools reveal deep insight into complex biology through an easy-to-use, intuitive interface.

Standard measurements are performed at room temperature. Optional accessories allow investigating liquids at low temperatures and – important for OLED applications – powders at high temperatures. The software offers various analysis functions, for example output of color coordinates. Two models of Hamamatsu Quantaurus-QY cover the wavelength range 300 nm to 950 nm (C11347-11) and 400 nm to 1100 nm (C11347-12).

The iQue Screener PLUS was created for researchers exploring the complex biology of the immune response. It offers all of the cost saving and productivity benefits delivered by the iQue Screener platform, plus more choice and flexibility. It features 15 detection channels, including 13 fluorescence and 2 scatter detectors, which provide for richer content through higher order multiplexing. Underneath the sleek exterior of the iQue Screener PLUS are high performance technologies designed to make it fast and capable, yet easy to use. By combining a powerful flow cytometry detection engine, with a novel and patented sample introduction mechanism, IntelliCyt has met throughput and content objectives in a way that no other suspension-cell-based screening platform can achieve. Bucher Biotec AG | www.bucher.ch

Visit us at MipTec

Labs using the Quantaurus QY often also have the Quantaurus Tau (C11367), which determines fluorescence and phosphorescence lifetimes down to sub-ns with photon counting sensitivity. Hamamatsu Photonics | www.hamamatsu.ch

Booth C34 / C41

Visit us at MipTec Booth A40

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Lifesciences plus 02 I 2015


Supporting your great ideas

Expertise in gas sensors

The World’s First Commercial Electrophoretic Tissue Clearing System The new X-Clarity Tissue Clearing System from the imaging specialists at Logos Biosystems provides an all-in-one solution for electrophoretic tissue clearing. X-Clarity Tissue Clearing System is based on the groundbreaking Clarity method, which provides unparalleled tissue samples for imaging. You can clarify a whole adult mouse brain in under 48 hours. Clarity is a novel technology that produces transparent tissues by forming a hydrogel network that supports the ultra- structure while allowing for the removal of lipids. Unlike other tissue clearing methods, the Clarity uniquely enables the efficient labeling of tissues with macromolecules such as antibodies and nucleic acids, making high resolution imaging possible and allowing to generate unprecedented 3D images that show the relationship between structure and function. Clarity overcomes the current limitation of light microscopy technology, which does not allow the imaging of tissues without first creating slices. The groundbreaking Clarity method was developed by the Deisseroth Lab at Stanford University (Chung et al, Nature 2013, 497:332-337) is trademarked to the Stanford University. Combining an innovative electrode design and an integrated cooling system, the X-Clarity Tissue Clearing System allows for faster and more consistent tissue clearing. Typical negative effects related with conventional electrophoretic tissue clearing methods include burning, melting, the formation of black precipitates, and bubble trapping. The main causes of these artifacts are due to irregular electric currents and poor temperature control during electrophoresis. The X-Clarity dramatically reduces the negative effects associated with standard electrophoretic tissue clearing with a unique electrode design and an active cooling system. A tissue sample is placed inside the X-Clarity ETC Chamber where it undergoes electrophoresis to remove lipids. Clearing buffer is continually circulated through the chamber under temperature controlled conditions, minimizing the probability of artifacts arising due to poor temperature control. Compared to other methods, the X-Clarity Clarity Tissue Clearing System dramatically reduces tissue clear- ing time. A whole adult mouse brain may be cleared within 48 hours. One of the most exciting features of the technology is that it allows for the penetration of and labeling with macromolecules such as antibod- ies or oligonucleotides. Now you can generate unprecedented 3D images from whole tissue that show the relationship between structure and function. Bucher Biotec AG | www.bucher.ch

Visit us at MipTec

FCX-MC25/MC95 series (oxygen)

Measuring range 0 % to 25 % or 0 % to 95 % High precision and fast response time Analogue or digital interface

EC4/SGX-4 series

Electrochemical 4 series (20 mm diameter) Measurement cells for industrial gases Suitable for portable measuring devices Long lifespan and low drift

Carbondio-ppm series (carbon dioxide) Measuring range 0 – 500 ppm up to 0 – 5000 ppm ± 2.0 % precision Response time (T90) adjustable from 1 s to 30 s Analogue or digital interface

MiCS MOS gas sensors

Low power consumption Ammonia, carbon monoxide, nitrogen oxide, ozone and VOC Available with evaluation electronics

Booth C34 / C41

Click and buy Lifesciences plus 02 I 2015

55

www.pewatron.com


BIOTECHNICA Three days. More than 600 exhibitors from 28 countries. A clear vision. A complete overview of the value chain across the whole biotech industry. Access to new contacts. International networking and partnering services. Staying up to date. Information and insights into Bioeconomy, Personalized Medicine Technologies and BioIT.

6 – 8 October 2015 Hannover ▪ Germany biotechnica.de fairs. tr ad e ket t. Two e ic k t ic A t IC O ne ECH N T s s IO e B c s ac Your rovide ION. also p T U L VO to L A B

IGZ Instruments at Miptec When Marcel Witzthum founded IG Instrumenten-Gesellschaft AG 50 years back in 1965, his aim was to supply the Swiss laboratory market with high quality analytical instruments. Half a century later, IGZ Instruments, with its three branches in Geneva, Fribourg, Basel and the main office in Zürich, has become a long term partner for many local laboratories. One of the secrets of IGZ is to import new and innovative products into the Swiss market. Today, the product portfolio covers Synthesis, Chromatography, Climate chambers, Freezers, Microbiology and last but not least, Life Sciences. At MipTec IGZ is present with its Chromatography and the Life Science products: QuickGene-Mini80 for DNA/RNA/Plasmid Isolation in 6 minutes This compact system requires no centrifugation step in the DNA/RNA isolation process from various samples. The QuickGene-Mini80 shares a common isolation mechanism and kits with a semi-automated nucleic acid isolation system and offers high performance at a reasonable price. The small, lightweight QuickGene-Mini80 uses a thin (80µm) porous membrane with outstanding absorptive and desorptive performances. High-purity nucleic acid can easily be obtained in high yield and at low pressure. Phos-tag for an easy and fast distinguishing of phosphorylated and non-phosphorylated proteins Phos-tag is a functional molecule that specifically binds all phosphorylated forms of Ser/Thr/Tyr. It is applicable for the specific separation of phosphorylated proteins as well as for western blot detection, purification by agarose gel chromatography and MALDI-TOF/MS analysis. IGZ Instruments AG | www.igz.ch Visit us at MipTec Booth D26

Handelskammer Deutschland-Schweiz verena.stuebner@handelskammer-d-ch.ch

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Supporting your great ideas

Expertise in pressure sensors Digital CMOS camera C11440-42U

Image Splitting Optics A12801-01

Dual Wavelength Imaging The ORCA-flash4.0LT + W-VIEW Gemini nicely complete each other to a dual wavelength imaging system for microscopy. The newly designed image splitting optics divides the field of view into two separate images and projects them side by side into the camera. When the camera is the ORCA-flash4.0-V2 or -LT, the image sensor’s internal electronic structure is – by architecture – also split into two halves. This synergy opens the use of innovative new functions like independent exposure times and subarrays for each half on the sensor. Exceptional QE across a wide range of wavelengths combined with low noise make the ORCA-flash 4.0LT a simple and affordable choice for low light imaging. With three times the field of view and two times the speed of traditional interline CCDs, this camera opens a host of new possibilities. Hamamatsu’s new W-View mode for the ORCA-flash4.0LT expands those possibilities even further. Dual wavelength florescence just got a lot less complicated. Simplifying multi-wavelength experiments like FRET, the W-VIEW Gemini / ORCA-flash4.0LT duo get out of your way to bring you closer to the biology. But what biological processes will this synergistic partnership bring closer to you?

AG/AP(B)/AL series From 0 – 20 mbar to 12 bar Analogue or digital ± 1.5 % precision FS

CCD54/CCD53 series From 0 – 2.5 mbar to 10 bar Analogue or digital ± 1.8 % to ± 2.2 % precision FS

Independent exposure times When imaging dual wavelengths in fluorescence, you very often face the situation of having very different light levels, resulting in suboptimal or even unusable pictures. The LT’s new W-VIEW mode takes away the need for complicated tweaks in optics or filtration by enabling individual exposure times for each wavelength. Problem

Solution

Exposure time

Exposure time

Exposure time

Exposure time

10 ms

10 ms

HPSD 3000/4000 series

Result

From 0 – 10 mbar to 7 bar Analogue and digital Integrated temperature sensor ± 0.7 % precision FS

50 ms (×5)

10 ms

Unbalanced

×5 long exposure

Balanced

exposure image

only for yellow

exposure image

Independent readout directions Some experiments require perfect synchronicity of the two image halves, which is by nature impossible to achieve with a single rolling shutter exposure sequence. The ORCA-flash4.0LT supports the necessary options to synchronize the shutters of the two image halves. Independent subarray Fast experiments can generate a lot of data. If you need to go fast but not necessarily keep every single pixel of the image, the option of using independent subarrays is the solution. High-speed, extended time course data are recorded with more simplicity than ever before. Hamamatsu Photonics | www.hamamatsu.ch

Stainless steel and ceramic pressure sensors From 0 – 20 mbar to 600 bar Analogue or digital Piezoresistive or capacitive

Visit us at MipTec Booth A40

Click and buy Lifesciences plus 02 I 2015

57

www.pewatron.com


Visit us at MipTec Booth A40

Hamamatsu FDSS MicroCell with EFS Head for In Vitro Toxicity Assays

H

amamatsu presents a very fast

model’ assays should possess higher

kinetic

significance for the targeted

fluorescence

reader

system based on the FDSS Mi-

application.

crocell, which is able to trace

spontaneous and electric field paced

All trends above target at a better out-

orescence can occur for example in car-

beating of cardiomyocytes, in particular

put of drug discovery pipelines of phar-

diomyocytes, which can show spontane-

for evaluation of cardiac side effects in

ma companies and biotech startups.

ous beating at 37 °C. The Ca2+ flux dur-

in vitro toxicology.

However, it turned out that one major

ing beating then leads to pulsed signals

drawback remains: the late appearance

synchronous to the beating of the cells.

In the past, screening campaigns for

of side effects of potential drug candi-

Cardiac side effects can easily be

new drugs in pharma industry were

dates. In particular, cardiac effects have

observed with this very fast kinetic

conducted by investigating several mil-

led to several major withdrawals of po-

reader system. Addition of drugs may

lions of compounds in search for poten-

tential drug candidates in late clinical

change the spontaneous beating fre-

tial hits. Today, strategies are changing

phases. The drug discovery world there-

quency, which can be measured with

substantially with the goal to improve

fore is looking out how side effects, in

highest sensitivity and accuracy. But

the output of the pharma R&D pipelines

particular cardiac ones, can be investi-

drug addition may also change the pulse

in terms of quantity and quality.

gated at a very early stage, best case ‘in

height or pulse shape of the beating (re-

vitro’ and on multi-well plate level.

lated to the action potential of the cells),

Trends to phenotypic screening allow a more focused pre-selection

it may lead to arrhythmia or to even to a

of compound candidates according

Hamamatsu offers a very suited solu-

complete stop of beating. All these re-

to prior knowledge.

tion for the needs of such in vitro toxi-

sponses are quantitative and reproduci-

Today, the rapidly growing under-

cology studies. Details are described be-

ble findings, which give significant indi-

standing of receptors, in particular

low. The Hamamatsu FDSS MicroCell is

cation for possible cardiac side effects.

of GPCRs, reaches down to the level

a kinetic fluorescence and lumines-

The measurement of beating over a

of their molecular structure. Compu-

cence reader for assay development and

whole multi-well plate with 120Hz and

tational simulation programs are used

small screens in 96- and 384-format.

several ten minutes delivers a huge

to evaluate specificity, reactivity and

Development of fluorescence over time

amount of measurement data. To ease

binding times of organo-chemical

can be measured for all wells in parallel

the challenge of data processing, Hama-

substances to the specific receptors.

as the reader is camera-based. For use

matsu Photonics offers a dedicated

Such simulation calculations can

in in vitro toxicology, this FDSS Micro-

‘Waveform Analysis Software for Cardio-

help confining the hit search by

cell reader can now be upgraded with

myocyte Beating’. It can be configured

screening to the most promising

2 dedicated extensions: the high speed

to determine frequencies and ampli-

classes of substances.

kit and the EFS head.

tudes, rising and falling slopes, and

Another big trend is taking advantage

The high speed kit makes use of a

other parameters with selectable win-

of stem-cell based assays: for example

very fast Electron Multiplication (EM)

dows in time and signal height and lim-

iPS derived cells can form good disease

Camera with high gain, which allows

its for detection of arrhythmia – all on

models by suited differentiation of

acquiring up to 120 frames per second

multi-well plate level.

stem cells into organ cells of interest.

for a 96 well plate. Thus very fast chang-

The beating of cardiomyocytes can

Screening hits done with such ‘disease

es can be observed. Fast changes of flu-

also be paced by the Electrical Field

58

Lifesciences plus 02 I 2015


different stimulation frequencies and at different electrical pulse conditions tell how well cells can be stimulated effectively. Any change of response to the electrical field stimulation due to addition of drugs may indicate a cardiac side effect. The big advantage of paced measurements is that data processing is definitely much easier compared to evaluation of traces with spontaneous beating, as the parameter frequency is no variable to be determined any more. Larger toxicology test series benefit

EFS Features

• 96-channel • Up to 120 Hz data acquisition • Compatible with heating 37 °C • Simultaneous injection and detection of all wells

Applications

• Cardiac toxicity of pharmacological compounds

• Pace beatings of cardiomyocytes • Phenotypic screening assays

from this method in terms of higher cal-

Hamamatsu Photonics

culation throughput.

www.hamamatsu.ch

Stimulation (EFS). This way spontaneous beating in the well is suppressed

Of course the High Speed Kit and the

and electrical field stimulation leads to

EFS Head can be used also for other ap-

synchronized beating of all cells. Hama-

plications* such as investigating stimu-

matsu Photonics has developed a dedi-

lation of neurons or of muscle cells.

The FDSS/µCell EGS system should not be used

cated so-called EFS Head, which re-

Thus, the EFS head option finds wide

for optically detecting/monitoring change in

places the dispenser head inside the

interest in toxicology screening as well

transmembrane potential of the cells. The FDSS/

FDSS Microcell. With this head it is pos-

as for basic research. For further read-

µCell EGS system should not be used on any

sible to synchronize the beating of cells

ing, please visit www.hamamatsu.ch >

cell or cells in which the user or anyone else has

inside each well. Response amplitude at

Drug Discovery Solutions.

expressed target ion channels.

*NOTE:

BD LSRFortessa™ X-20 with BD reagents

THE VERY BEST FOR INSTRUMENTAL TLC

A brilliant new approach to multicolor cell analysis on the benchtop.

TLC VISUALIZER PROFESSIONAL VISUALIZATION, DOCUMENTATION AND EVALUATION OF HPTLC AND TLC PLATES

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WORLD LEADER IN PLANAR CHROMATOGRAPHY

Becton Dickinson AG Binningerstraße 94 4123 Allschwil Switzerland Tel.: +41 61 485 22 22 infoch@europe.bd.com www.bdbiosciences.com/eu BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. ©2015 BD. A084-00


IMMUNOLOGY

PD-1/PD-L1 Blockade Bioassay for Therapeutic Antibodies in Development for Immunotherapy Currently, methods to measure the potency of drugs targeting PD-1 or PD-L1 include in vitro binding assays, primary T cell-based cytokine release assays, and in vivo model systems. A novel bioluminescent reporter assay that can be used for rapidly quantifying function of PD-1 or PD-L1 therapeutic antibodies in development as measured by activation of NFAT signaling pathway has been developed.

MEI CONG, ZHI-JIE JEY CHENG,

and characterization in early drug dis-

NATASHA KARASSINA, JAMISON GRAILER,

covery, and for lot release and stability

Bioassay Specificity and Suitability for Monitoring Antibody Stability

JIM HARTNETT, NEAL COSBY AND FRANK FAN

studies in drug manufacture.

We have developed PD1/PD-L1 block-

I

mmunotherapy enables a patient’s own immune system to battle cancerous

target

cells. Programmed

Improved Bioluminescent Reporter-Based PD-1/PD-L1 Blockade Bioassay

ade bioassay into a thaw-and-use format, and the cell batches are functionally tested. The Thaw-and-Use PD-1/ PD-L1 bioassay is convenient, and

death receptor-1 (PD-1), serving as

We developed a bioluminescent report-

exhibits assay specificity [Figure 2A].

a negative costimulatory receptor on T

er assay that can be used for rapidly

No premixture of antibody drug and

and B cells, binds to its ligand PD-L1

quantifying function of PD-1 or PD-L1

cells is required for efficient blockade.

(B7-H1), which is aberrantly expressed

therapeutic antibodies in development

Our data demonstrate that the PD-1/

on a wide array of human cancers. En-

as measured by activation of NFAT sig-

PD-L1 blockade bioassay can be used

gagement of PD-1 by PD-L1 expressed

naling pathway. We engineered a Jurkat

to measure the antibody potency of ei-

on tumor cells disables the host antitu-

T-cell line stably expressing NFAT-

ther anti-PD-1 or anti-PD-L1. The assay

mor response and the effect is associated

luciferase reporter and human PD-1

is specific as demonstrated by a non-

with poorer prognosis of the patient.

(PD-1 effector cells). Cells stably ex-

related antibody, anti-CTLA-4 drug Ip-

PD-1 and PD-L1 are important targets

pressing human PD-L1 and a TCR acti-

ilimumab, that failed to activate the

for cancer immunotherapy. Investigation-

vator can act as antigen presenting cells

NFAT pathway.

al anti-PD-1 immunotherapy drugs, such

(PD-L1 aAPC cells). Three steps of sig-

as nivolumab from BMS and lambroli-

naling pathway alterations representing

Many human monoclonal antibodies

zumab (MK-3475) from Merck, demon-

the antibody’s mechanism of action are

display poor biophysical properties in-

strate significant overall survival rates in

achieved in this single-step, homogene-

cluding sensitivity to pH and UV light,

patients with advanced melanoma.

ous cell-based assay [Figure 1]. Co-

low stability, and a propensity to aggre-

Currently, methods to measure the

cultivating the two cell lines induces ac-

gate. Here we demonstrate that the

potency of drugs targeting PD-1 or

tivation of the Jurkat NFAT pathway via

PD-1 Blockade Bioassay is suitable for

PD-L1 include in vitro binding assays,

antibody crosslinking of T-cell receptor

detecting potency changes of stressed

primary T cell-based cytokine release

(TCR) activator/TCR complex. PD-1

antibody samples [Figure 2B and C].

assays, and in vivo model systems.

signaling in PD-1 effector cells follow-

Compared to antibody stored at 4  °C

There is urgent need for a simple, ro-

ing engagement of PD-L1+ cells inhibits

as positive control, test antibody #1

bust, plate-based functional bioassay

T cell function, and results in NFAT

completely lost its activity after one day

to measure potency of a drug candidate

pathway inhibition. Blockade of PD-1/

of heat-stress treatment at 65 °C, where-

that blocks the interaction of PD-1/

PD-L1 interaction with either anti-

as potency of test antibody #2 was right

PD-L1 for drug discovery and develop-

PD-L1 or anti-PD-1 mAbs can reacti-

shifted, indicating that test antibody

ment. Such an assay requires high sen-

vate the NFAT pathway in a dose-

#2 is more resistant to heat treatment at

sitivity with appropriate specificity, pre-

dependent manner.

65 °C.

cision, and accuracy for drug screening

60

Lifesciences plus 02 I 2015


01

 Figure 1: Three major events occur in the Promega homogeneous PD-1/PD-L1 Blockade Bioassay. Event 1: TCR-mediated NFAT activation occurs when PD-1 Effector cells and PD-L1 aAPC cells are engaged through TCR/TCR activator interaction. Event 2: Inhibition of NFAT signal by PD-1/PD-L1 ligation when no blocking antibodies are present. Event 3: Recovery of NFAT signal by addition of anti-PD-1 or anti-PD-L1 blocking antibody.

02

Figure 2A: Assay specificity of PD-1/PD-L1 Blockade Bioassay: Serial dilutions of anti-human CD274 (B7-H1, PD-L1) antibody (BioLegend, Cat.# 329709), anti-PD-1 antibody (BioLegend, Cat.# 329911), or Ipilimumab (Yervoy, BMS) were added into co-culture system of PD-1 effector cells and aAPC PD-L1 cells, incubated for 6 hours at 37 °C before luciferase activity was quantified using Bio-Glo Reagent. Data was fitted using 4PLC curve fit of GraphPad Prism software. Figure 2B and C: Anti-PD-1/PD-L1 blocking antibody #1 and #2 were heated stressed at either 42 °C or 65 °C for indicated times before being applied to cellbased PD-1/PD-L1 Blockade Bioassay.

Bioassay Qualification and Tolerance to Human Sera Bioassay qualification was performed

assay is a good candidate for further de-

AUTHORS

velopment into a NAb assay. Mei Cong, Zhi-jie Jey Cheng, Natasha Karassina,

to demonstrate that the PD-1/PD-L1

Summary

Blockade Bioassay is able to measure

The Promega PD-1/PD-L1 Blockade

Frank Fan. Promega Corporation, Mei Cong, PhD,

relative potency for antibody biologics

Bioassay is designed to reflect mode of

Director fo CAS, R&D, mei.cong@promega.com,

with good linearity, accuracy, precision,

action with great assay specificity, re-

www.promega.com

and reproducibility, from qualification

peatability and linearity. The assay can

assays run at four different test anti-

be used to detect antibody stability. As

body potencies (range 50 – 200 %) rela-

the assay can tolerate human sera, fur-

tive to 100 % reference sample across

ther development of the system into a

three different assay days (data not

neutralizing antibody detection (NAb)

shown). The assay was also challenged

assay is feasible. 

Jamison Grailer, Jim Hartnett, Neal Cosby and

with various amounts of human sera

Join the talk by Dr. M. Cong on Reporter Bioassays at #MipTec during the Industry Symposium on September 22.

from 1 % to 10 % in assay buffer. Resulting EC50s and fold inductions of test antibody were minimally impacted by human sera, suggesting that the bio-

Lifesciences plus 02 I 2015

61

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F I LT R AT E I Jo b O p p o r t u n i t i e s

NEWS

Roche to acquire Kapa Biosystems to strengthen next-generation sequencing product offerings

Roche

announced that it has signed a definitive agreement to acquire Kapa Biosystems, Inc. (Kapa), a privately-held company headquartered in Wilmington, Massachusetts, US. The transaction is subject to customary closing conditions. Kapa Biosystems is a provider of genomic tools in the life sciences sector that employs proprietary technologies to optimize enzymes for next-generation sequencing (NGS), as well as polymerase chain reaction (PCR) and real-time PCR applications. Kapa’s proprietary protein engineering technology is highly customizable and allows for the generation and screening of large numbers of enzyme variants. Tailored enzymes with improved performance for specific applications can be rapidly selected, expediting product development timelines. Kapa’s impressive portfolio of NGS reagents includes enzymes such as novel DNA polymerases, with the potential to improve the performance of the entire sequencing workflow. “This acquisition builds on Roche’s commitment to develop a differentiated NGS portfolio that will provide our customers with a complete genetic testing solution,” said Roland Diggelmann, COO Roche Diagnostics Division. “Kapa’s technology and products complement our current expertise and offerings such as the portfolio of target enrichment products for NGS.” “Joining Roche provides us access to their broad product portfolio, global reach and clinical expertise that will accelerate our strategy of offering comprehensive NGS workflow solutions to more laboratories around the world,” said Paul McEwan, Co-Founder and Chief Scientific Officer of Kapa Biosystems. “We are also excited to have the opportunity to further leverage our enzyme engineering capabilities with the ultimate goal of having a more significant impact on medicine and human health.” #  www.roche.com

Aerial view of Roche Basel from south with river Rhine, Switzerland. (Picture: Roche)

Drosophila melanogaster, fruit fly, is a common model organism for studies in embryogenesis and cell biology. (Picture: Wikimedia)

Omya ist ein führender, globaler Hersteller von Industriemineralien auf der Basis von Calciumcarbonat und Dolomit sowie weltweit in der Distribution von Spezialchemikalien tätig. Gegründet 1884 in der Schweiz, ist Omya heute mit über 8‘000 Mitarbeitenden in über 50 Ländern tätig. Infolge Pensionierung des Stelleninhabers suchen wir für unser Verkaufsteam einen dynamischen und kompetenten

Divide and rule: a tumour’s strategy

When normal

Sales Manager Life Sciences Switzerland (m/w)

body cells escape the control from their peers, a tumour can form and eventually lead to cancer. The team of Eduardo Moreno, professor at the Institute of Cell Biology at the University of Bern, has discovered that a mechanism that is known from the early development of embryos plays a role in the earliest stages of adult tumour development. As part of their SNSF-funded project, the researchers were able to film the cells of developing fruit fly pupas, which carried an artificially activated gene called Myc. The gene alone was sufficient to induce abnormal cells to divide more actively, squeeze through between healthy cells, kill them and take over their place. This represents an unexpected mechanism of invading tissues in the first phase of tumour development. This invasion mechanism is known to be active during embryonic development when cells rearrange themselves to transform the body shape. “Divide and rule”, the famous military strategy, is how the researchers describe the aggressive cells’ behaviour. The mechanism could explain the earliest beginnings of tumour development of most cancer types and is different from invasion mechanisms of metastases in later phases.

Aufgaben • Akquisition und Verkauf des Produktportfolios mit Umsatz- und Ergebnisverantwortung • Mitwirken bei der Umsetzung der Lieferantenstrategie in der Omya Organisation • Beziehungspflege mit Kunden und Lieferanten • Mitwirken im Business Development • Marktbeobachtung (Marktentwicklung, Mitbewerber, Umwelt etc.) Anforderungsprofil • Technische Grundausbildung (Chemie) mit Weiterbildung im kaufmännischen Bereich • Fundierte Fachkenntnisse im Bereich Kläranlagen, Wasseraufbereitung und Kehrichtverbrennung • Mehrjährige Berufserfahrung und Leistungsausweis im Verkauf • Persönlichkeit mit unternehmerischem Denken und Handeln • Sehr gute Sprachkenntnisse in Deutsch und Englisch (jede weitere Sprachen ist von Vorteil) • Reisebereitschaft

About 90 percent of all cancers form in lining tissues (epithelia) like the one filmed in the pupas: in colon, skin or the mammary gland. The manipulated Myc gene is the most commonly misregulated gene in tumours. The identified mechanism could therefore apply to many cancers and help scientists to find new strategies to prevent tumour formation at its root before much damage has been caused. #  www.snf.ch

Omya (Schweiz) AG I Frau Brigitte Stampfli Baslerstrasse 42 I CH-4665 Oftringen Tel. +41 62 789 2307 I Mail jobs.ch@omya.com

62

Lifesciences plus 02 I 2015


Functional Drug Screening Systems

Scientific Cameras

(FDSS)

spontaneous beating

W-VIEW GEMINI • Matched to the performance of Gen II SCMOS Cameras • Chromatically corrected • User-defined filter combinations • Easely aligned and stable • High transmittance

Option: Electric Field Stimulation (EFS) Head for Cell-based Assays

ORCA-Flash4.0 LT • USB 3.0 connectivity • W-View mode • Versatile • Large dynamic range

EFS Features: • 96-channel • Up to 120 Hz data acquisition • Compatible with heating 37°C • Simultaneous injection and detection of all wells

ImagEM X2 SERIES • Extreme low-light performance • Fast frame rates • Long exposure capability • High-speed readout

Applications: • Cardiac toxicity of pharmacological compounds • Pace beatings of cardiomyocytes • Phenotypic screening assays

ORCA-Flash4.0 V2 • Large dynamic range • Quantitative • Fast frame rates • Light-sheet readout mode

Note: The FDSS/μCELL EFS system should not be used for optically detecting change in transmembrane potential of the cells, and should not be used with the cells in which the user or somebody else expressed target ion channels.

FDSS MicroCell 96/384

swiss@hamamatsu.ch – Tel. +41 32 625 60 60 – www.hamamatsu.ch Lifesciences plus 02 I 2015

with 1.0 Hz stimulation

63


F I LT R AT E I N e w s

How zebrafish rebuild the skeleton of amputated fins

The zebrafish

Bone formation upon partial amputation of zebrafish tail fins. L: normal regeneration of fin rays; R: irregular bone formation with widely expanded production of the signalling protein Sonic Hedgehog. (Picture: Developmental Biology, University of Bayreuth)

(Danio rerio) is a popular ornamental fish. When parts of its tailfin are injured by predators, or are experimentally amputated, the lost tissue is replaced within three weeks. Prof. Dr. Gerrit Begemann, at the Developmental Biology unit at the University of Bayreuth and Ph.D. student Nicola Blum published new results about how zebrafish manage to regenerate the exact shape of the lost fin skeleton in two articles in the journal “Development.” In order to rebuild an amputated fin, a large number of new osteoblasts have to be formed by cell divisions from existing osteoblasts. For this to happen, osteoblasts exposed to the vicinty of the wound have to abandon the production of bone material and revert, or dedifferentiate, to a “rejuvenated” developmental stage. This switch requires retinoic acid levels to drop below a critical concentration. However, upon amputation the tissue beneath the wound initiates a massive bout of retinoic acid synthesis that is required to mobilize cell division in the fin stump. Blum now found out that Osteoblasts that participate in regeneration transiently produce Cyp26b1, an enzyme that destroys and inactivates retinoic acid. Protected by this process, osteoblasts are able to turn into precursor cells that contribute to a pool of undifferentiated cells, the blastema. Cells in the blastema then provide the building blocks for the regenerated fin. Some parts of the blastema, which replenish the supply of cells needed for regeneration to occur, continue to produce retinoic acid. This allows two processes to run in parallel during regeneration: Proliferation for the production of all cells that replace the lost structure and redifferentiation of osteoblasts where the skeleton re-emerges. In a second study Blum broke down the events required for skeletal pattern regeneration. The local production of a signalling protein called Sonic Hedgehog pilots immature osteoblasts to areas where existing fin rays are to be extended. Signal production only occurs in locally restricted cells that are free of retinoic acid. Such epidermal cells produce Cyp26a1, an enzyme that is functionally similar to Cyp26b1. #  www.uni-bayreuth.de

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