Optimize, Adapt and Overcome - 18

Optimize, Adapt, and Overcome: Tips from the Cell Line Development Front * Confident Assurance of Clonality Using Calcein AM with Minimal Effect on Viability

To establish that the level of staining is sufficient to detect the majority of cells, we counted the total number of void wells, single-cell
wells and multiple-cell wells for all the 3 concentrations of CAM. Under 5 µM CAM, 58.2% of
the wells were devoid of cells, whereas 31.3%
contained a single cell, and 10.5% (n=380
wells) contained multiple cells. Meanwhile under 1 µM CAM staining levels, 58.7% of wells
contained no cells, 30.3% contained a single
cell, and 11.1% (n=760 wells) contained multiple cells. Finally, under 0.5 µM CAM staining
levels, 96.6% of the wells contained no cells,
2.1% of the wells contained a single cell and
1.3% (n=760 wells) of the wells contained multiple cells. Comparing the cell per well valence
distribution with theoretical values for a 0.5
cell per well limiting dilution shows that the 5
µM and 1 µM staining conditions are very similar to the predicted values, whereas those of
0.5 µM differ significantly (Figure 3G). A quantification of the difference shows that the 5 µM
and 1 µM conditions diverge by <5% whereas
that of 0.5 µM diverges by >90% from the predicted values (Figure 3H).
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Using the White-Light workflow to
assess the effect of CAM on viability
Fluorescent probes that stain living cells are notorious for having deleterious effects on cell viability. As such, we sought to test the effect that
CAM has on outgrowth of clones after limiting
dilution. We evaluated colony outgrowth from
the cells that were stained with 0.5 µM, 1 µM,
and 5 µM CAM prior to limiting dilution. To control for this process, a similar batch of cells was
mock-stained using vehicle prior to setting up a
limiting dilution in identical fashion. The plates
were scanned using white light on the CSI on day
0 and a number of subsequent days (days 1, 2,
3, 6) until they formed prominent colonies (day
10). Representative plates of clonal outgrowths
for each condition at day 10 are shown in Figure
4A. A quick comparison of day 10 colonies shows
that the 5 µM condition (Figure 4A, lower right
panel) has far fewer colony outgrowth compared
to other staining conditions and label-free control. We tallied the number of wells that formed
single-colonies for each condition. The label-free
control derived colonies showed 26.3% ±1.9 (n
= 1196 wells) single-colony outgrowth. Mean-

while 29.6% ± 3.2 (n=760 wells) and 30.1% ± 3.5
(n=760 wells) showed single-colony outgrowth
for the 0.5 µM and 1 µM staining condition. In
contrast, only 10.3% (n=380 wells) of wells grew
single-colonies for the 5 µM staining condition, a
2.5-fold decrease (Figure 4B). This decrease in the
percentage of outgrowth is consistent with cytotoxicity of CAM at higher concentrations, which
result in a dramatic effect on proliferation from
single cells. In comparison, the 0.5 µM, 1 µM, and
label-free control show no discernible differences
in the number of raw single-colony outgrowth.
Another observation is that the colonies derived
from 5 µM staining appear to be smaller compared with colonies derived from label free or
lower CAM concentrations. Representative day-6
colonies from each condition are shown (Figure
4C). A quantification of surface area of day 6 colonies shows that 5 µM CAM-stained cells are ~40%
smaller compared with their label-free counterparts. Meanwhile, there was no measurable difference in colony size between 0.5 µM and 1 µM
CAM-stained colonies and the label-free control.
Therefore, the use of CAM dye at a high concentration

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