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FONDAZIONE FILARETE STEM CELL PLATFORM SERVICES

Stem cell platform wants to offer specific services in order to assist and train other researchers to approach protocols and techniques peculiar of haematopoietic and muscular stem cells areas. In particular we propose: •Production and distribution of proliferation and differentiation media, specific for stem cell culture •Stem cells isolation and supply •Basic cell culture training (cell lines)

•Advanced cell culture training (stem cell isolation and culture; primary cell culture preparation) •Cytometric characterization of cell samples •ELISA tests •Viability and cloning tests

Fondazione Filarete Viale Ortles 22/4 20139 – Milano (MI) - Italy Phone +39 02 566601 Fax +39 02 537250 Email services@fondazionefilarete. com


PROLIFERATION and DIFFERENTIATION MEDIA Production and distribution of specific media for stem cell culture (proliferation and differentiation).

Transfer of know how and results from research to create a product helpful also for future clinical applications in stem cell field

Laboratory equipment: •Class 2 microbiological safety cabinet •Fluorescence inverted microscope (Leica DMI 4000B) Local Contacts: Marzia Belicchi (marzia.belicchi@fondazionefilarete.com) Silvia Erratico (silvia.erratico@unimi.it)


STEM CELL ISOLATION and SUPPLY

Laboratory equipment: •MACS Miltenyi Biotech for immunomagnetic sorting (LS columns) •Inverted microscope (Leica DM IL)

Microscope technical characteristics: •Samples are focused with the quadruple objective nosepiece. •The transmitted light illumination allows the integration of the contrast slide (for phase and modulation contrast) •The fluorescence filter cubes perform a matched combination of excitation, reflection, band-pass and barrier filters.

Non adherent

Applications: Immunomagnetic sorting allows to purify a particular cell subpopulation thanks to beads coniugated antibodies. A tested protocol for isolation, created specifically to obtain a high level purity, consents to isolate also a low-expressed cell populations, such as CD133+ progenitors, derived from different tissue (for example blood or muscle) Microscope visualization is essential to control morphology and cell culture progression. Adherent Local Contacts: Marzia Belicchi (marzia.belicchi@fondazionefilarete.com) Silvia Erratico (silvia.erratico@unimi.it)


CELL CULTURE TRAINING

Laboratory equipment: •MACS Miltenyi Biotech for immunomagnetic sorting (LS columns) •Thermo Scientific Heracell 150i incubator (CO2 and O2 control) • Stereomicroscope (Leica L2) •Inverted microscope (Leica DM IL)

•Class 2 microbiological safety cabinet •Horizontal laminal air flow cabinet

Applications: This service is conceived to help researchers to approach cell culture and train them to gain specific skills and techniques. Two approaches are thought up: • A basic cell culture training: for acquiring the fundamental skills with cell lines • An advanced cell culture training: focused on more difficult techniques, such as stem cells isolation and culture or primary cell culture preparation (for example, human or mouse muscle tissue explants) Local Contacts: Marzia Belicchi (marzia.belicchi@fondazionefilarete.com) Silvia Erratico (silvia.erratico@unimi.it)


CYTOMETRIC CHARACTERIZATION of CELLS

Laboratory equipment: cytofluorimeter Cytomics FC500 Beckman Coulter Technical characteristics: LASERS: • Uniphase Argon ion, 488nm, 20mW output • Coherent Red Solid State Diode, 635nm, 25mW output FLOW CELL: •150 × 450 micron rectangular channel BioSense •enhanced optics quartz mounted with vertical (upwards) flow path for superior hydrodynamic •Focusing DETECTORS: • Forward Scatter Detector – Solid state detector with Patented Fourier design providing integrated dual collection angle selection. • Side Scatter Detector – High performance photodiode • Fluorescent Detectors – High performance photomultipliers with spectral sensitivity from 185nm-900nm Local Contacts: Marzia Belicchi (marzia.belicchi@fondazionefilarete.com)


STEM CELLS REFERENT SCIENTIST Prof. Yvan Torrente: Neurologic Sciences Department, Università degli Studi di Milano (yvan.torrente@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Stem Cells Platform is able to grow several human stem cell lines and possesses patents for stem cell culture on both serum-based and serum-free media. The facility aims at developing a unique know-how for the culture of human stem-cells and eventually for full scale production of clinical-grade human stem cells.

PLATFORM CAPABILITIES The following services can be activated under specific request: Cell delivery: • human and mouse adult stem cells of different tissue origin (peripheral blood, muscle, bone marrow, neural, mesenchymal) • Specific stem cell populations isolated from pathological tissue • from dystrophic muscles (Duchenne muscular dystrophy, Becker muscular dystrophy, shoulder joint) • from brain tumors like glioblastoma multiforme The platform routinely delivers the following cell types:

• human CD133+ stem cells isolated from blood • human CD133+ stem cells isolated from muscle • human mononuclear • human and mouse myoblasts (wild type and dystrophic mdx and scid/mdx mice) from primary cultur • human and mouse fibroblasts from primary cultures • mouse C2C12 myoblast lines • mouse 3T3 fibroblast lines • cancer stem cell from human glioblastoma • clones obtained from cancer stem cells of human glioblastoma Validation of muscular regeneration and migratory abilities of human stem cells in an immunocompromised dystrophic animal model. Staminality tests: • self-renewal: percentage of primary colonies able to originate secondary colonies • Clonogenic ability • Differentiative capabilities: endothelial, hematopoietic, muscular Cytogenetic tests: • up-regulation of oncogenes • telomer length • telomerasic activity


STEM CELL SERVICES SERVICES

Short, 1-day, training in cell cultures (per person)* Long, 4-days, training in cell cultures (per person) * 2 days training in stem cells (per person)* 2-days training in primary cultures (per person)* Growth medium for AC133 stem cells from blood (100 ml) Growth medium for AC133 stem cells from muscle (100 ml) Differentiation medium for AC133 stem cells from blood (100 ml) Differentiation medium for AC133 stem cells from muscle (100 ml) Cytofluorimetry (excluding reagents) (per hour) ELISA tests (excluding reagents) (per test) Karyotype (per test) Inverted Microscope with LED Illumination Leica DM IL LED (per hour)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com


REFERENCES Colleoni F, Torrente Y. The new challange of stem cell: brain tumor therapy. Cancer Letters (2008) Meregalli M, Farini A, Torrente Y. Combining stem cells and exon skipping strategy to treat muscular dystrophy. Expert Opin. Biol Ther 8(8), 1051-61 (2008) Marchesi C, et al. Correlation of circulanting CD133+ progenitor subclasses with a Mild phenotype in Duchenne Muscular dystrophy patients. PLoS ONE 3 (5) (2008) Cassano M, et al. Magic-Factor 1, a Partial Agonist of Met, Induces Muscle Hypertrophy by Protecting Myogenic Progenitors from Apoptosis PLoS ONE 3 (9) (2008) Marchesi C et al. Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection. Glia 55(4), 425-38 (2007) Torrente Y et al. Autologous Transplantation of Muscle-Derived CD133+ Stem Cells in Duchenne Muscle Patients Cell Transpl. 16(1), 1-100 (2007) Torrente Y Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 1, 1-13 (2007)

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