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Application note Proliferation & morphological parameters

Assessing proliferation directly in cell culture vessels without markers Background

Optimizing cell proliferation is an important part of in vitro cell culture. Using the right medium as well as the best coating allows for obvious improvement in cell growth and general culture health. Allowing cells to grow as uninhibited as possible lessens the selective pressure that otherwise may result in a culture that differs too much from the original cells. This in turn allows for more reliable experimental results and the possibility to detect smaller differences with statistical significance. In vitro cultures should preferably be kept in their exponential growth phase to reduce the probability of cell line changes caused by e.g. long-term growth inhibition due to confluent cultures. The possibility to quickly and non-invasively obtain an objective value on the growth state of a cell culture could thus improve the quality of routine as well as experimental cell culturing.

often rely on a dye to make the cells visible and attached cells must be detached To make it more convenient to count numerous parallel cultures, assays using 96-well plates have been developed. Using a specific stain, they measure an intracellular process or an intracellular content, e.g. mitochondrial activity (MTT, Alamar Blue), number of lysosomes (Neutral red uptake) or the cellular amount of proteins, DNA or RNA. It is assumed that the amount of dye bound in the cells corresponds to the cell number. A treatment that disrupts the specific cellular mechanism which is the target of the assay may yield unreliable results. To have reliable results, it is essential to use two assays with fundamentally different endpoints such as mitochondrial activity and protein measurements.

Previously radioactive labels have commonly been used for cell proliferation studies. The labels often targeted cellular DNA, RNA or proteins. The amount of radioactivity detected in the cells was assumed to correspond to the cell number in the sample. The above mentioned methods have in common that they affect or even destroy the cells during the counting process. This makes it necessary to seed parallel cultures for every timepoint studied in the proliferation experiment. To measure cell proliferation with HoloMonitor™ M3 there is no need to detach or in any way affect the cells. The cultures studied using the HoloMonitor™ M3 are not adversely affected and can be used for further experiments. Also, using HoloMonitor ™ M3, the same cells in the same cell culture vial can be

End-points

Cell proliferation can be measured using several different types of endpoints. The most straight forward method is to simply count the cells. Cells either grow attached or as suspension cells. They can, using a grid in a microscope, be counted directly. A more common method is to transfer a small aliquot of cells to a counting chamber, such as a haemocytometer, and then count the cells in a microscope. This requires chemical or physical detachment of the attached cells. To make the process of cell counting faster, several instruments that count cells automatically, such as coulter counters and particle counters, have been developed. These instruments

Figure 1. Screenshot of the HoloStudio™ 2.1 software of reconstructed cells for further analysis of parameters like cell number, confluence, total phase shift.

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Application note

used for several weeks of investigation, thus allowing for long term studies of cell proliferation. Holomonitor ™ M3 counts the cells in the culture, but also gives values for confluence and cell dry mass. Thus any of these three parameters can be used for proliferation studies.

Application

To perform a proliferation study using the HoloMonitor™ M3 the sample is placed on the objective table. The study can then be performed either as a time-lapse study, in which case A

B

C

D

the cells should be put on a heating stage or in a micro-incubator, or at 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 (Mölder et al., 2008). Reconstructed holographic 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 shape, size and thickness can be measured. The HoloMonitor™ M3 allows users to capture both holographic and phase-contrast images. The holographic images are then segmented. When settings have been decided for the first image, the rest of the images in the series are segmented accordingly. The data is then presented in one of several common spreadsheet programs on a cell-to-cell basis for easy data analysis. Not only do the data include parameters that are important in regard to proliferation but also morphological data that show morphological changes in a culture over time.

Preliminary results

Figure 2. Counting yields results equivalent to counting in a haemocytometer (A and D). Using the HoloMonitor™ M3 to measure cell confluence (B) yields results comparable to cell counting. Instead of confluence, cell dry mass can be used as a cell counting parameter (C).

As has been shown previously (Mölder et al., 2008), using the HoloMonitor™ M3 for cell counting yields results equivalent to counting in a haemocytometer (Fig. 2 A and D). Using the HoloMonitor™ M3 to measure cell confluence (Fig. 2 B) yields

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results comparable to cell counting. Instead of confluence, cell dry mass can be used as a cell counting parameter (Fig. 2 C). Confluence measurements or cell dry mass measurements can be put to good use for cell cultures where the cells are growing on top of each other, and therefore are difficult for the cell counting program to separate out.

Discussion

Cell counting with HoloMonitor™ M3 has been proven to be as accurate as the standard cell counting method using a haemocytometer (Mölder et al., 2008). The HoloMonitor™ M3 produces not only cell numbers, but also an array of additional data such as cell confluence, cell dry mass, cell size and cell shape. This new noninvasive and nondestructive way to measure the proliferation of the cells can replace any method currently used. It requires no extra chemicals, cultures or incubation periods, and the cells can be counted in almost any cell culture vessel. As the same culture can be reused for measurements day after day, only a limited number of replicate cultures are needed. Also, the cultures used for the proliferation studies can afterwards be used for other assays as the cells are not affected by the counting procedure. References 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. Journal of Microscopy 232, 240-247, 2008


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