Scale down of the cytotoxicity assay volume to increase compound

We designed, optimized and successfully automated the fluorescent protein based cytotoxicity assay for 96 well plates, which was followed by primary and confirmatory compound screening of a small compound library (for the statistical characterization of the assay in 4.1.3.8, we used the control wells of the plates from the experiments presented later in point 4.3.4). In order to increase the assay throughput to screen larger compound libraries, we implemented the miniaturization of the cytotoxicity assay for 384 well plates. Preliminary experiments during optimization was carried out by using the mCherry expressing Mes-Sa cell line.

Learning from the previous optimization procedure, we checked firstly the 2D distribution of cells. The wells on a 384 well plate have a different geometry: in contrast to the circular wells of a 96 well plate, wells of the 384 well plate are square shaped with rounded corners. Not surprisingly, also on the 384 well plates cells tend to migrate and grow close to the wall of the wells after attachment. We observed also intraplate differences. Cells in wells that are in the middle of the plate proliferated by the well’s wall in equal distribution (the gradient of cell mass was increasing to every direction from the middle of the well equally), while cells that were seeded in a side well (that is by the edge of a plate) migrated more likely to that wall or corner of the well, which is not in connection with other wells (Figure 25). As the middle of the wells were almost

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unpopulated in every part of the plate, we set a 4 point scanning protocol (4x250 flashes) with 1.75 mm distance between the points, where the highest cell density was expected (the diameter of the well is 3 mm).

A)

B)

Figure 25. A) Mes-Sa mCherry cells with various initial cell no. imaged by a fluorescent microscope after 72 h incubation. Red, green and blue rectangles refer to the intraplate position of the imaged wells. Effect refers to the observed gradient of cell mass. B) Heat map of Mes-Sa mCherry cells in the 4 corner wells (marked with blue in panel A), scanned and visualized by the Perkin EnSpire microplate reader.

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In the next step, we optimized the initial cell number. Theoretically, the optimal initial cell number for Mes-Sa mCherry would be 1250 cells/well, as the area of one well on the 384 well plate is approx. ¼ of the area of a well on a 96 well plate, where we seeded 5000 cells/well. Thus, we examined the growth characteristic of 3 different initial cell numbers, 625-, 1250-, and 2500 cells/well. When half amount of the expected initial cell number was seeded (625 cells/well), the confluency of the well after 120 h was still very poor, that is apparent also from the RFU values (Figure 26/A). Interestingly, when 1250 cells/well were seeded, cell density was still not sufficient, as seen at 72 h, and the growth did not start to saturate until 120 h. The 2500 cells/well setting returned a better confluency after 72 h, and we observed that the growth followed the well-known logistic equation.

For the initial cell number optimization, cells were seeded and followed in 60 μl of medium. We investigated also, if the change in the final volume is influencing cell growth. Thus, we designed a plate layout, where 2500 cells/well (Mes-Sa mCherry) were kept in 40-, 60-, or 80 μl of medium. By following cell growth for 120 h, we didn’t notice any difference in the proliferation rates (Figure 26/B). For practical reasons, we decided to keep using the 60 μl of final volume.

However, what we observed during these preliminary experiments was the non-negligible effect of the evaporation of medium from the 384 well plate, especially from the outer rows and columns of wells, which was visible also by eye. Thus, we compared cell growth also by dividing the microplate into 3 parts: (1) the outer wells, which are on the edge of the plate, (2) the wells in the second rows/columns and (3) inner wells (the rectangle defined by the wells C3-N3-C22-N22). The growth rate was influenced by the extent of evaporation, as both the outer and the second row/column wells suffered variable levels of decrease in growth rates compared to the inner wells (Figure 26/C). As both medium evaporation and uneven distribution of the cells were more prominent in the outer wells, we excluded the outer and second line wells from further experiments, in favor of smaller interplate differences, and better assay robustness. However, as a positive control (dead cells), we used the second line wells in certain plate layouts (see in Appendix 2).

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Figure 26. Growth kinetics of Mes-Sa mCherry cell line under different conditions in 384 well (‘w’) plates. A) Influence of the initial cell number to cell growth kinetics. B) Cell growth in different volumes of medium. C) Effect of the evaporation of medium to the growth.

0 24 48 72 96 120

C) Effect of medium evaporation on growth

time (h)

B) Effect of the volume of cell culture medium on growth

time (h)

A) Effect of the initial cell number on growth

time (h)

RFU

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After cell growth measurements, we followed the same logic as earlier, and checked the growth inhibition of the test compound NSC693871. The growth inhibition was followed daily for 144 h. At each day, the fluorescent intensity values were normalized to the control wells (complete inhibition and untreated control), and GI50 values were obtained (Figure 27).

Figure 27. Cytotoxicity of a test compound (NSC693871) against Mes-Sa mCherry on a 384 well plate, measured daily for 144 h. GI50 values (in μM) were obtained based on sigmoidal curve fitting, standard deviations (sd) were calculated from 8 parallel experiments.

As a conclusion, we were able to demonstrate the dose-dependent cytotoxicity of NSC693871, thus mCherry fluorescence based assay was successfully optimized for 384 well plates.