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Application note Proliferation & morphological parameters

Assessing morphological changes in cell cultures Background

Changes in shape or structure of a cell is often caused by intra or extracellular conditions. During regular cell growth conditions there is an ongoing change in morphology due to the motility of the cells and because of cell cycle variations. One of the more extreme morphological changes a cell goes through, especially adherent cells, is the mitosis. Starting out flat, widely spread, irregular and often elongated, the cell turns into a thick, circular object with a small area during mitosis. As the cells divides there is also a halving of the volume. A normal cell culture thus contains a certain amount of morphological variance, even if all cells in the culture are identical regarding cell type and proliferation capacity. Thus, small changes in cell morphology may be disguised in the normal morphological variation between cells in a culture. In contrast, a growth-inhibited culture often display a more homogeneous morphology as cells no longer loosen and round up in order to divide.

curately. All measurements are relative and depend on the focusing.

Application

To measure cell morphology changes using the HoloMonitor™ M3, samples are placed on the objective

A

B

End-points

Morphology today is most commonly measured on light microscopy images using various software solutions that analyze two-dimensional phase contrast images. There are several commonly used analysis programs [1]. The drawback of these analysis programs is that it is not possible to quantify phase contrast images ac-

C

Figure 2. Morphology analysis of L-929 cells analyzed daily 1-7 days after seeding. The x-axis represents the time after seeding and the y-axis represents the morphological parameters in arbitrary units. (A) represents the average area per cell, (B) represents the thickness per cell, (C ) represents the volume over time per cell. Control sample (blue), substance A and B (green and dark red) and 2% and 5% DMSO (orange and yellow).

Figure 1. Morphology of are analysis as they are segmented in the HoloStudio™ software.

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Application note Figure 3. Cell number of L-929 cells analyzed daily 1-7 days after seeding. The x-axis represents time after seeding in days and the y-axis represents the average number of cells in each image. Control sample (blue), substance A and B (green and dark red) and 2% and 5% DMSO (orange and yellow).

table. The study can then be performed either as a timelapse 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. Each picture is reconstructed into 3D representations of the sample as previously described [2]. These images are segmented to provide morphological data on a cell-to-cell basis. Reconstructed phase images (Fig. 1) provide the starting point for further image analysis. The data available after segmentation show general proliferation data such as confluence and cell number as well as morphological parameters such as area, thickness and volume. The thickness is an estimation of optical path length. Morphology changes in a cell line can easily be quantified using data provided by HoloMonitor™ M3. As the data from each time-point come from all cells in the images captured at that time-point, the analysis results in a statistically significant dataset. Each and every cell contributing to the data can be followed back to the original image to guarantee the accuracy of the region settings.

Results

The experiment was set up to investigate the morphological effects of several substances on the mouse fibroblast cell line L929. Images of

the cell cultures were captured every day for 7 days with 10-15 images per culture at each time-point. DMSO was used as a positive control at 2% and 5%. We also used two unknown substances, A and B. Figure 2A shows how the average area per cell varied as the experiment proceeded. The area of the control cells and cells treated with substance A was consistent throughout the experiment while DMSO treatment caused the cells to have larger areas. Substance B caused the cells to shrink. Substance B actually killed the cells by day four of treatment Cellular thickness was consistent in control cells and cells treated with substance A (Fig. 2B). DMSOtreated cells became thinner and substance B-treated cells became thicker. Cellular volume decreased the first three days of seeding in control and substance A-treated cells and then was stable (Fig. 2C). DMSO and substance B treatment clearly decreased the cell volume. Morphological changes can clearly be seen day two after seeding in cells treated with substance B or DMSO. When looking at the average number of cells per image, there is no clear difference in cell number compared to control until day three after seeding. This suggests that morphological differences can be observed prior to any effect on cell growth. Figure 3. Cell number of L929 cells analyzed daily 1-7 days after seeding. The x-axis represents time after

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seeding in days and the y-axis represents the average number of cells in each image. Control sample (blue), substance A and B (green and dark red) and 2% and 5% DMSO (orange and yellow).

Discussion

Morphological changes, for whatever reason they may occur, can be detected by the HoloMonitor™ M3 on a cellular basis. The morphology is represented by parameters such as area, thickness and volume. Regardless of what type of cell culture experiment the HoloMonitor™ M3 is used for, cells can always be non-destructively and non-invasively analyzed for morphological differences. This means that users gain an increased amount of data during each experiment without needing extra culture flasks or parallel cultures. Depending on the circumstances this may enable faster experiments with a lower threshold of detection. References http://www.cellprofiler.org 2010 01 15 http://www.valasciences.com/software/id/ cyteseer/ CyteSeer software 2010 01 15 http:// www.moleculardevices.com/pages/software/ metamorph.html 2010 01 15 http://www.mediacy.com image; web.uvic. ca/ail/techniques/imagepro.html 2010 01 15 www.Zeiss.com 2010 01 15 Mölder, A., Sebesta, M., Gustafsson, M., Gisselson, L., Gjörloff-Wingren, A., Alm, K. Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography. Journal of Microscopy 232, 240-247, 2008


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