Optimize, Adapt and Overcome - 13

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

count feature was used automate to cell counts.
Plates were subsequently scanned using only the
brightfield standard resolution scan mode on
days 0, 1, 2, 3, 6 and 10.
Calculating viability
To quantify raw outgrowth, single-colony wells
were counted on day 10 and displayed as a percentage of the total wells. Single colony-containing wells were then evaluated on a well-by-well
basis for the presence of single cells on day 0 to
determine clonal outgrowth, displayed as the
percentage of single cells that subsequently
formed colonies.

Instrument Overview and Workflow
Confirming that a cell type expressing a biologic
originated from a single cell progenitor is a critical requirement for cell line development and
regulatory approval. Two general imaging approaches can be used to determine whether a
cellular population is monoclonal in origin. The
first approach assesses cells in a label- free fashion, whereas a new emergent method uses fluo13

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rescence to detect the presence of cells. Both
approaches involve a sequence of acquisition
analysis and reporting steps.

The label-free approach to
assessing monoclonality
A standard limiting dilution of 0.5 cells per well
is expected to yield 30% single cell-containing
wells, however identifying those single-cell wells
at day 0 is difficult. Debris and well artifacts can
easily be mistaken for cells, making the process
subjective and error-prone. As a result, plates are
typically scanned in white light repeatedly over
the course of several days, beginning with day 0,
to monitor the growth of cells into colonies (Figure 1 , left column). Once prominent colonies have
formed (usually within 6-10 days), they can be
tracked back to day 0 to determine the presence
of a single cell. This process can be time-consuming, but the Loci Count feature on the CloneSelect Imager helps improve this process by automating the detection of single colonies (yellow
box) and linking various time points of a plate
scan. Meanwhile, the Report Generator feature
allows the identification of cell regions (green box

in Figure 1 ) and debris regions (not shown), and
the exporting of a monoclonality report for submission to regulatory agencies. Wells where single colonies form and can be shown to originate
from a single cell are designated as monoclonal
and carried forward to assess productivity (titer)
and scale-up.

The fluorescence approach to
assessing monoclonality
An alternative approach to the assessment of
monoclonality involves using fluorescence, a
powerful biological tool that generates high signal-to-background (Figure 1 , right column). One
method involves using a fluorescent probe to
stain cells expressing the biopharmaceutical target protein of interest. The stained cells are then
seeded into microplates using a limiting dilution
approach. Scanning a plate on the fluorescencecapable CSI allows acquisition of high-resolution
images in fluorescence and white light. One advantage of using fluorescence is that plates can
be analyzed for clonality immediately after seeding (on day 0). In comparison, the white light
workflow requires waiting until prominent

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Optimize, Adapt and Overcome

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