High Efficiency Single Cell Cloning of hiPSCs While Ensuring Maintenance of Phenotype

Drug discovery, synthetic biology and cell therapy efforts are increasingly using human induced pluripotent stem cells (hiPSCs). However, the inability to robustly manipulate hiPSCs as single cells has remained a significant biological and technical hurdle. Current techniques rely on inefficient methods such as limiting dilution (LD), colony picking and fluorescence-activated cell sorting (FACS), which are time consuming, expensive and incompatible with the sensitivity of hiPSCs. Furthermore, these methods provide insufficient documented evidence of clonality. This application note describes how several different hiPSC lines have been successfully subcloned in a robust and automated fashion using the VIPS instrument in conjunction with optimized culture conditions.

This method has been shown to reduce time and cost while most importantly maintaining the cell phenotype and genomic integrity as well as providing documented evidence of cell line clonality. Firstly, we show that the VIPS plus MatriClone, as a platform combination, results in a 3- to 4-fold improvement in clonal colony outgrowth of single seeded hiPSCs when compared with manual LD. Secondly, three hiPSC subclones derived from four cell lines, with presumed healthy or disease-affected backgrounds, were selected for expansion and extended characterization following VIPS seeding and whole well imaging. Expression of pluripotency was confirmed for each of the sub cloned lines. Finally, we demonstrate that the subclones maintained key hallmarks of hiPSCs, including genomic integrity and stability after extended culture and successful neuronal differentiation to demonstrate potency.