WEBINAR | Establishing New Standards in Efficient Single Cell Cloning of hiPSCs
Tuesday, September 22, 2020 | 11am EDT (NA) / 4pm BST (UK) / 5pm CEST (EU-Central)
Drug discovery, diagnostic, and therapeutic efforts are increasingly using human-induced pluripotent stem cells (hiPSCs) and related technologies. 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), which are time consuming, expensive, incompatible with the sensitivity of hiPSCs, and ultimately, ineffective in addressing concerns about clonality. For example, hiPSC gene-editing efficiencies can be low, thus requiring the generation and screening of hundreds of clones, which in turn must be validated for clonal origins.
At Solentim, we have previously established the benefits of our proven VIPS™ commercial single cell seeding platform over manual LD for the creation of master cell banks, which helped shape industry standards for clonality of therapeutic CHO and HEK cell lines. Until now, the technology has been incompatible with hiPSCs due to their inherent sensitivity and particular culture needs, such as specialized pre-plated matrices and daily feeding schedules. Here we present a robust and clinically relevant workflow for the single-cell subcloning of hiPSCs using the VIPS seeding platform in combination with a new soluble dispensing matrix, MatriClone™(Solentim). MatriClone is an animal component-free matrix dispensed at the time of seeding, which allows for cell attachment and growth without the need for pre-coating culture plates. The VIPS instrument is uniquely able to perform the dual functions of high efficiency single-cell seeding and whole-well imaging to document outgrowth and verify clonal origin. The combination of this instrument and dispensing matrix provides a novel solution for improving workflows for cell reprogramming or gene editing of hiPSCs.
In this webinar, we demonstrate that numerous hiPSC cell lines can be successfully subcloned in a robust and automated fashion using the VIPS instrument in conjunction with optimized culture conditions, thereby reducing time and cost while most importantly maintaining the integrity of clonal biology. Firstly, we show that the VIPS plus MatriClone combination results in a several-fold improvement in clonal colony outgrowth of single seeded hiPSCs when compared with manual LD. Secondly, a number of hiPSC subclones were identified from presumed healthy and disease-affected backgrounds using daily whole well imaging on the VIPS and selected for expansion approximately 10-14 days post-seeding. Expression of pluripotency was confirmed for each of the sub-cloned lines. Finally, we demonstrate that subclones that maintained genomic integrity after extended culture and successfully differentiated into mixed cortical neurons.
The robustness of this workflow has immediate potential to impact standards, consistency, and confidence of clonality in both academic and pharma research. Furthermore, future translation of this workflow could positively effect GMP standards and re-establish expectations with the regulatory bodies for the development and production of advanced cell models, cellular diagnostics, and cell therapeutics, including clonality documentation to accompany future IND submissions.