Application Note

Development Of A Highly Efficient, Documented Workflow For Making Clonal Cell Banks Of Gene-Edited iPSCs

By Philip D. Manos, Evercel and Ian Taylor, Duncan Borthwick, and Claire Richards, Advanced Instruments

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Over the last decade, laboratories engaged in therapeutic monoclonal antibody production have engaged a ‘clonally- derived’ methodology which seeds individual cells from pools of cells thereby limiting heterogeneity. This practice, moreover, requires quality evidence of clonality, supporting the existence of a single cell post dispensing, for submissions to the medicines agencies.

Laboratories involved in cloning human induced pluripotent stem cells (hiPSC) cells will be familiar with the practice of limiting dilution (pipetting cells at a calculated <1 cell per well concentration) or using platforms such as fluorescently activated cell sorting (FACS). However, these methods are generally inefficient (low cell survival and low outgrowth) and moreover, do not support image-based evidence for purposes of assurance. In the case of FACS, this is also not conducive with a manufacturing environment.

Gene-editing steps in the process add additional challenges. Gene-editing methodologies often compromise cell health and survival. Depending on the complexity and type of approach utilized, this can dramatically change the scale of the project to find ultimately, a small number of cells that have survived the process, grown into colonies and then have, post genetic analysis, been found to contain the desired combination of edits.

We have previously reported on using a VIPS™ (Verified In- situ Plate Seeding) instrument (Solentim) and MatriClone™ a laminin-based matrix (Solentim) in a workflow for single cell seeding with parental hiPSC cell lines. VIPS offers both automated seeding and supporting clonality assurance through on- board imaging. With the additional numerical challenges of gene-editing workflows, we employed the same methodology here to examine the effectiveness of the VIPS + MatriClone workflow for gene-edited hiPSC cells.

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