Optimize, Adapt and Overcome - 20

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

results in a dramatic decrease in clonal outgrowth
and smaller colonies compared with label- free
control.

Using the combined WL and FL workflows
to assess the effect of CAM on viability of
colonies derived from single cell wells
So far we've discussed the viability of clones at
the population level. We used this simple approach in order to optimize the concentrations
of CAM to test. However, this data does not directly measure the viability of single cells. Hence,
we wanted to confirm that the observed singlecolony outgrowth percentage is truly accurate by
looking at colony formation derived from single
cells on a well-by-well basis. This was accomplished by calculating the percentage of singlecell wells that were able to form a colony. From
this more direct comparison, 75% ±8 (n=1520
wells) of single-cell wells form colonies in the 1
µM CAM staining condition. Similarly, 73.6% ±9.2
(n=1196 wells) of single-cell wells form colonies
for the label-free control. Meanwhile, a mere
21.4% of single-cell wells form colonies for the 5
µM CAM staining condition (Figure 4E). The con20

| GENengnews.com

sistency of raw outgrowth of single colonies and
colonies from single-cell wells shows that there is
no effect on cell viability using the CAM viability
dye at the 0.5 or 1 µM staining concentrations.

Guidelines on optimizing calcein AM
dye concentrations
An ideal concentration of dye balances the ability
to detect a majority of labeled- cells with minimal
cytotoxic effects on clonal outgrowth. We tested
a few concentrations, namely 0.5, 1, and 5 µM, for
the viability dye calcein AM. We found 1 µM to be
the ideal concentration based on the detection
and viability readouts. A similar assay optimization step would need to be performed based
on the dye, cell type, imaging system, and other
experimental conditions.

Conclusion
Traditionally, the process of establishing a clonal
cell line for use in the manufacturing of monoclonal antibodies has avoided the use of fluorescence reagents because of concerns over cytotoxicity and genetic alterations that may occur.
The development of inexpensive and

accessible next-generation sequencing technologies though has alleviated many of the concerns
surrounding genetic alterations, providing an
opportunity to use fluorescence dyes if the issues surrounding cytotoxicity can be overcome.
Here, we demonstrate an optimized workflow
using the fluorescence reagent, calcein-AM, in
conjunction with a fluorescence-capable CSI that
shows similar viability to label-free conditions
while simultaneously providing high assurance of
clonality. In this workflow, we found that a 1 µM
concentration of calcein-AM is ideal for detection
of single cell on the CSI while not inducing any
cytotoxicity.
The benefit to using a fluorescence approach
for confirming monoclonality is that it allows for
the automated detection of single cells, significantly reducing the number of hours required to
manually identify single cells. The analysis can
be immediately performed on day 0 as well, providing information on clonality much earlier in
the workflow. As with any approach, there are
trade-offs to using fluorescence. Most obvious,
it requires additional sample prep and finding

http://www.GENengnews.com

Optimize, Adapt and Overcome

Table of Contents for the Digital Edition of Optimize, Adapt and Overcome