Application note

changes in cell cultures

Proliferation & morphological parameters

Assessing morphological

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

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).

BackgroundChanges in shape or structure of a cell is often caused by intra or ex-tracellular conditions. During regular cell growth conditions there is an ongoing change in morphology due to the motility of the cells and be-cause of cell cycle variations. One of the more extreme morphological changes a cell goes through, espe-cially adherent cells, is the mitosis. Starting out flat, widely spread, ir-regular 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 halv-ing of the volume. A normal cell cul-ture 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 be-tween cells in a culture. In contrast, a growth-inhibited culture often dis-play a more homogeneous morphol-ogy as cells no longer loosen and round up in order to divide.

End-pointsMorphology today is most common-ly measured on light microscopy im-ages 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-

ApplicationTo measure cell morphology chang-es using the HoloMonitor™ M3, sam-ples are placed on the objective

curately. All measurements are rela-tive and depend on the focusing.

A

B

C

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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).

Application note

table. The study can then be per-formed either as a timelapse study, in which case the cells should be put on a heating stage or in a micro-in-cubator, or as separate time-points after treatment, in which case no special equipment is necessary. Im-ages 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 quanti-fied using data provided by Holo-Monitor™ 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.

ResultsThe experiment was set up to inves-tigate 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 im-ages per culture at each time-point. DMSO was used as a positive control at 2% and 5%. We also used two un-known substances, A and B. Figure 2A shows how the average area per cell varied as the experiment proceeded. The area of the con-trol cells and cells treated with sub-stance A was consistent throughout the experiment while DMSO treat-ment caused the cells to have larg-er areas. Substance B caused the cells to shrink. Substance B actually killed the cells by day four of treat-ment Cellular thickness was consis-tent in control cells and cells treated with substance A (Fig. 2B). DMSO-treated cells became thinner and substance B-treated cells became thicker. Cellular volume decreased the first three days of seeding in con-trol and substance A-treated cells and then was stable (Fig. 2C). DMSO and substance B treatment clearly decreased the cell volume. Mor-phological changes can clearly be seen day two after seeding in cells treated with substance B or DMSO. When looking at the average num-ber of cells per image, there is no clear difference in cell number com-pared to control until day three after seeding. This suggests that morpho-logical differences can be observed prior to any effect on cell growth. Figure 3. Cell number of L929 cells analyzed daily 1-7 days after seed-ing. The x-axis represents time after

seeding in days and the y-axis repre-sents 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).

DiscussionMorphological changes, for what-ever 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. Regard-less of what type of cell culture exper-iment the HoloMonitor™ M3 is used for, cells can always be non-destruc-tively and non-invasively analyzed for morphological differences. This means that users gain an increased amount of data during each experi-ment without needing extra culture flasks or parallel cultures. Depending on the circumstances this may en-able faster experiments with a lower threshold of detection.

Referenceshttp://www.cellprofiler.org 2010 01 15http://www.valasciences.com/software/id/cyteseer/ CyteSeer software 2010 01 15 http://www.moleculardevices.com/pages/software/metamorph.html 2010 01 15http://www.mediacy.com image; web.uvic.ca/ail/techniques/imagepro.html 2010 01 15www.Zeiss.com 2010 01 15Mölder, A., Sebesta, M., Gustafsson, M., Gissel-son, L., Gjörloff-Wingren, A., Alm, K. Non-inva-sive, label-free cell counting and quantitative analysis of adherent cells using digital hologra-phy. Journal of Microscopy 232, 240-247, 2008

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