Automated selection and harvesting of pluripotent stem cell colonies

Abstract

The ability of pluripotent stem cells to differentiate into specialized cells of all three germ layers, their capability to self-renew, and their amenability to genetic modification provide fascinating prospects for the generation of cell lines for biomedical applications. Therefore, stem cells must increasingly suffice in terms of industrial standards, and automation of critical or time-consuming steps becomes a fundamental prerequisite for their routine application. Cumbersome manual picking of individual stem cell colonies still represents the most frequently used method for passaging or derivation of clonal stem cell lines. Here, we explore an automated harvesting system (CellCelector™) for detection, isolation, and propagation of human embryonic stem cells (hESCs) and murine induced pluripotent stem cells (iPSCs). Automatically transferred hESC colonies maintained their specific biological characteristics even after repeated passaging. We also selected and harvested primary iPSCs derived from mouse embryonic fibroblasts expressing the green fluorescent protein (GFP) under the control of the Oct4 promotor using either morphological criteria or GFP fluorescence. About 80% of the selected and harvested primary iPSC colonies gave rise to homogenously GFP-expressing iPSC lines. To validate the iPSC lines, we analyzed the expression of pluripotency-associated markers and multi-germ layer differentiation potential in vitro. Our data indicate that the CellCelector™ technology enables efficient identification and isolation of pluripotent stem cell colonies at the phase contrast or fluorescence level.