Methods for Automated Single Cell Isolation and Sub-Cloning of Human Pluripotent Stem Cells

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell−derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications. © 2020 The Authors.

Basic Protocol: Efficient automated hPSC single cell seeding and clonal expansion using the iotaSciences IsoCell platform

Alternate Protocol 1: hPSC single cell seeding and clonal expansion using the Cellenion CellenONE single-cell dispenser

Alternate Protocol 2: hPSC single cell seeding and clonal expansion using the Cytena single-cell dispenser

Support Protocol 1: Coating cell culture plates with Geltrex

Support Protocol 2: hPSC maintenance in defined feeder-free conditions

Support Protocol 3: hPSC passaging in clumps

Support Protocol 4: Laminin 521 coating of IsoCell plates and 96-well/384-well plates

Support Protocol 5: Preparation of medium containing anti-apoptotic small molecules

Support Protocol 6: 96- and 384-well target plate preparation prior to single cell seeding

Support Protocol 7: Single cell dissociation of hPSCs

Support Protocol 8: IsoCell-, CellenONE-, and Cytena-derived hPSC clone subculture and expansion