Application note Proliferation & morphological parameters
Assessing acute or long-term toxicology without markers Background
Toxicology is the science of evaluating the potentially adverse effects of substances. The research field contains numerous categories such as chemical, environmental and forensic toxicology. In vitro or cellular toxicology, is often used to evaluate the adverse effects of various substances. The LD50 value, the dose at which 50% of the test subjects are dead, is a very basic measure of the toxicity of a substance. This test was usually performed on test animals. With the introduction of in vitro cell cultures, which became widespread during the 1980s , the tests are performed much easier and result in EC50 values instead. The EC50 value is the concentration that results in 50% of the measured effect, e.g. 50% inhibition of cell growth. Furthermore, in vitro cell cultures provide the tools to extract the mechanism of action of the potential toxin.
Today, endpoints for cell culture experiments are often designed for evaluation in 96 well plates. These
multi well plates allow for a rather fast evaluation of several concentrations in parallel. The draw-back is that for every concentration and every time-point separate wells are needed. The measuring of the endpoint almost always requires the sacrifice of the cell culture. The end point usually is the amount of proteins or DNA in the cells or lysosomal or mitochondrial activity. These endpoints are used to determine relative amount of cells in the wells. HoloMonitorTM M3 can determine the cell proliferation by cell counting, confluence measurements or determination of cell dry mass without affecting the cells in any way. It also allows for quantitative determinations of cell morphology, making morphology changes accessible for toxicity evaluations. Cultures studied using the HoloMonitorTM M3 are not adversely affected and can be used for further experiments. It is quite challenging to evaluate the effect of the long-term versus acute toxicity of a substance. Using HoloMonitorTM M3, the same cells in the same cell culture vial can
be used for several days and even weeks of investigation, thus allowing for studies of both the acute effects and the long term effects using the same cell culture.
To investigate the effects of a substance on an in vitro cell culture the sample is placed on the objective table of the HoloMonitorTM M3. The
Figure 2. The effect of 10 () or 50µM (p) etoposide treatment compared to control () as measured using the HoloMonitor™ M3.
Figure 1. Screenshot of the HoloStudio™ 2.3 software of reconstructed and segmented cells for further analysis of parameters like cell number, confluence, total phase shift.
study can then be performed either as a time-lapse study, in which case the cells should be put on a heating stage or in a micro-incubator, or as separate time-points after treatment, in which case no special equipment is necessary. Images are captured automatically or manually, as set by the operator. After completion of the image capture, the images are processed and reconstructed as previously described . Reconstructed phase images (Fig. 1) provide the starting point for further image analysis. Cell number as well as confluence and total phase shift can be determined. The total phase shift corresponds to the total dry mass of the cells in the image. Further parameters like cell size and thickness can be measured. The HoloMonitorTM M3 is able to capture images from almost all standard cell culture containers and does not hinder or affect the possibility to use other methods
as end-points after capturing holographic images. The HoloMonitorTM M3 will allow a user to extract more data from each experiment for a negligible additional amount of time and with no extra material required.
When using the HoloMonitorTM M3 to study the cell number of etoposide treated cells it is clear that the highest dose of etoposide, i.e. 50 μM, resulted in cell growth inhibition (Fig. 2). The lower etoposide concentration, i.e. 10 μM, at first resulted in a stimulation but later in an inhibition of growth. Confluence measurements show the same results. Interestingly, the 10 μM etoposide treatment results in thicker cells, but at the end of the study all cells were clearly thinner than in the beginning. Control cells had a larger cell area than etoposide-treated cells.
0 µM etoposide
When using the HoloMonitorTM M3 to study proliferation or any other proliferation-related effect such as LC50, morphological parameters can also be studied. To extract these extra
50 µM etoposide
Figure 3. When using the HoloMonitorTM M3 for toxicology studies the results include not only cell proliferation and confluence, but also quantifiable morphological parameters such as cell thickness and area.
data during a toxicology experiment is very valuable for the analysis of the in vitro cell cultures. Depending on the nature of the morphological changes it may also contribute to the understanding of the mechanism of action for the substance. Each captured image contributes to building a statistically sound basis for each time-point instead of relying on parallel cultures where the cells have to be trypsinized for each time-point when growth is to be measured. As each image potentially contains hundreds of cells, analysis will provide a large amount of information, making it possible to detect minor morphological changes between each time-point as well. References  Zucco, F., De Angelis, I., Testai, E., Stammati, A. Toxicology investigations with cell culture systems: 20 years after. Toxicology in Vitro 18, p. 153-163, 2004.  Mölder, A., Sebesta, M., Gustafsson, M., Gisselson, L., Gjörloff-Wingren, A. and Alm, K. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography. J Microscopy 232, 240-247, 2008