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

Abstract

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 subcloning 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.

High‐throughput and automation advances for accelerating single‐cell cloning, monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

“…cellenONE® has 2 holders for microplates, which can be used as either a source and/or a target; its ability to automatically and sequentially load one sample after another into the dispenser head makes it an ideal system for high‐throughput, non‐supervised, fully‐automated cloning with very high clonal cell outgrowth rates (https://www.cellenion.com/). Vallone et al successfully used the cellenONE® platform to isolate single‐cell clones with a normal karyotype from a heterogeneous human pluripotent stem cell (hPSC) population 45 …”

“…The single cell‐derived hPSC sub‐clones from each system maintained pluripotency and genetic stability. Among the three instruments, isoCell showed the highest cloning efficiency after the single‐cell deposition step 45 …”

Microfluidics on Standard Petri Dishes for Bioscientists

“…Grids and circuits have now been used for all core cell‐culture methods (e.g., feeding, [ 15 ] replating, [ 15 ] cloning, [ 15,23 ] and cryopreservation) [ 15 ] with organisms ranging from bacteria [ 15 ] through yeast [ 24 ] and worms [ 15 ] to man, [ 14–15,23 ] as well as for drug screening, [ 14–15 ] chemotaxis [ 14 ] plus cell‐wounding assays, [ 25 ] lysis plus RT‐PCR, [ 14 ] transfection plus genome editing, [ 15 ] and fixation plus immunolabeling. [ 15 ] Note that although most of these applications are cell‐based, the general technology can nevertheless be applied wherever small volumes require manipulation.…”

The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions

“…In brief, small guide RNA (sgRNA) targeting the M2 gene close to the mutation site to be corrected were the transfection we analyzed the bulk population using the Sanger sequencing to estimate the editing efficiency. Thereafter, the automated single cell cloning of the genome edited cell pool was performed as described in protocol 42 . The clones were screened by SANGER sequencing.…”