Poster from the Barcelona Meeting in June 2015. Shows the following key features: 1. For some cell types, semi-solid can be useful to keep cells from moving during outgrowth 2. Cell Metric can clearly image single cell in SSM on day of seeding 3. Cell Metric can be used for single round of cloning with 3 month time saving in cell line development

1. Assessing Clonality of Production Cell Lines
via High-Resolution Imaging
Melisa Carpio, Bhavya Kadambi, Elizabeth Stangle, and Sanjay Patel
Takeda California, 10410 Science Center Drive, San Diego, CA 92121, USA
ABSTRACT
BACKGROUND AND NOVELTY:
Demonstrating that a production cell line originated from a
single clonal progenitor is a regulatory requirement prior to
commercial approval. Most approaches to meet this
requirement rely on theoretical calculations involving probability
distributions, cell plating densities, and numbers of colonies
observed. Based on the specifics of the subcloning procedure,
however, a 2nd round of cloning is typically also needed to
provide conservative assurance of clonality. Unfortunately, this
2nd round of cloning is laborious and adds up to three months to
cell line development timelines. In an attempt to streamline this
process, we have evaluated the use of the Cell Metric to
provide high-resolution, single cell images that can be used to
prove clonality with just one round of subcloning.
EXPERIMENTAL APPROACH:
Initial tests using our CHO cell lines demonstrated that the Cell
Metric yielded high quality images capable of easily
discriminating cells from debris present in both the culture and
the plate. Subsequent optimization work involving inoculation
density and culture media studies were performed to identify
conditions favoring growth of a colony from a single cell in each
well. In the course of this work, we observed a considerable
amount of cell migration within each well on a day-to-day basis
when using liquid media. As a result, we shifted our focus to
plating in the cells in a semi-solid methylcellulose media.
RESULTS AND DISCUSSION:
Using the Cell Metric with our optimized methylcellulose plating
method, we were able to clearly identify single cells within a well
and track their growth at the same location until the colony
reached confluence. In this manner, the entire history of
candidate production cell lines could be documented and traced
back to a single clonal progenitor. Taken together, our
evaluation strongly suggests the Cell Metric is capable of
providing direct evidence of monoclonality with one round of
subcloning, potentially reducing cell line development timeline
by up to three months.
(1) THE CELL METRIC IMAGER
(4) MEDIA OPTIMZATION TO IMPROVE COLONY GROWTH
a) b)
SUMMARY
 Be able to clearly track growth of a single
cell to confluence
 Semi-solid media is being used
 Work is currently on-going to comprehensively validate the system and reduce the occurrence of false negatives
 High quality images
• Automatic focusing
• Direct visual reading of whole well
• Unlimited number of data points
• Digital record of cells
 High-throughput
• 10 plate incubated stacker
• 3-4 minutes per plate
Figure 1: The Cell
Metric CLD System
(2) UNDESIRABLE CELL
MIGRATION IN LIQUID MEDIA
 Images of single cells are clearly observed but cells tend to
migrate during daily handling
Day 0
Day 1
Day 0
Day 1
Figure 2: Examples of cell migration from Day 0 to Day 1
 Commercially available methylcellulose was imaged
 Significant debris was present in three of the four media
 Semisolid #4 was chosen for further optimization
Figure 3: Images of the four types of methylcellulose
(3) SEMI-SOLID MEDIA SCREEN
Semisolid #1 Semisolid #2
Semisolid #3 Semisolid #4
 The problem of cell migration was solved by using semi-solid media but poor growth was observed with no wells reaching confluence (Figure 4)
Figure 4: Images showing one cell not growing past a few doublings
Day 0 Day 1 Day 2 Day 3
 A media optimization experiment was done to optimize colony growth
• A matrix of two media (Media #1 and Media #2) and three additives
(Additive #1, Additive #2a, and Additive #2b) was tested with duplicate
96WP seeded at 1 cell/well for each media and additive combination
 Figure 5 has plate maps for each of the media conditions at Day19
• Media #1 is the negative control and again showed no colonies
• Media #2 shows an improvement in cell growth
• Both additives enhanced growth for both types of media with Additive #2
yielding more colonies than Additive #1
 For all media conditions, the number of colonies originating from 1 cell/well
was sub-optimal (<50% of the total colonies; Table 1)
Media #1 Media #1 + Additive #1
Media #1 + Additive #2a Media #1 + Additive #2b
Media #2 Media #2 + Additive #1
Media #2 + Additive #2a Media #2 + Additive #2b
Media Condition
Colonies from
1 cell/well
Colonies from
>1 cell/well
Edge + False
Negatives
Media #1 0 1 0
Media #1 + Additive #1 5 4 1
Media #1 + Additive #2a 9 8 0
Media #1 + Additive #2b 7 9 0
Media #2 5 11 2
Media #2 + Additive #1 14 15 5
Media #2 + Additive #2a 14 27 13
Media #2 + Additive #2b 12 41 4
Table 1: Average number of colonies observed in a 96WP at Day19
Figure 5: Day 19 plate maps for each of the media conditions
(5) FINDING AN OPTIMAL SEEDING DENSITY TO TARGET 1 CELL/WELL
1.0 cell/well 0.75 cells/well 0.50 cells/well 0.25 cells/well
Seeding Density
(cells/well)
Colonies from
1 cell/well
Colonies from
>1 cell/well
Edge + False
Negatives
1.0 21 23 13
0.75 17 17 10
0.50 15 9 6
0.25 11 4 2
Table 2: Average number of colonies observed in a 96WP at Day16
Figure 6: Day 16 plate maps for each of the plating densities
 Duplicate 96WP were seeded at 1.0, 0.75. 0.50, and 0.25 cells/well
 As expected, fewer colonies were present as the plating density
decreased (Figure 6)
 Both 0.50 and 0.25 cells/well had >50% of the colonies coming from
1 cell/well (Table 2)
 Determine media conditions that fosters
growth of single cells
 A Media #2 and additive combination was
identified
 Reliably deposit single cells into a single
well of a 96WP
 Lowering the cell density increases the
number of colonies coming from 1 cell/well
(6) EXAMPLES OF TRACKING GROWTH FROM A SINGLE CELL TO CONFLUENCE
Day 0 Day 1 Day 2 Day 8
Day 0 Day 1 Day 5 Day 19
a) c)
b) d)
Figure 7: Tracking growth from a) 1 cell/well, b) >1 cell/well, c) a false negative, and d) the edge of the well
Day 0 Day 1 Day 2 Day 5
Day 0 Day 2 Day 8 Day 16
 Figure 7 shows that some wells are easy to analyze while others are more difficult, making validation challenging