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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 2 nd round of cloning is typically also needed to provide conservative assurance of clonality. Unfortunately, this 2 nd 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
