Methods for automated single cell isolation and sub-cloning of human pluripotent stem cells


  • V.F. Vallone
  • N.S. Telugu
  • I. Fischer
  • D. Miller
  • S. Schommer
  • S. Diecke
  • H. Stachelscheid


  • Current Protocols in Stem Cell Biology


  • Curr Protoc Stem Cell Biol 55 (1): e123


  • 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.