Discussion

Pluripotent stem cells (PSCs) and primary cells are foundational tools for basic research and applied applications including regenerative therapy, drug discovery, and toxicological assessment. While stem cells have a tremendous proliferative capacity, long term culture of these cells has been shown to cause an accumulation of mutations that result in genetic instability, increasing tumorigenicity and thus limiting their usefulness in research and clinical applications. Improved solutions for cryopreservation of early passage cells that minimize loss of viability, maximize post-thaw recovery, and minimize unwanted differentiation are essential components to PSC, as well as primary cell, workflows. While many cryopreservation reagents afford high viability immediately post-thaw, significant apoptosis and necrosis is often observed following the first 24 hours post-thaw, decreasing the effective viability, reducing cell numbers and adding additional stress and selective pressure to cultures. Further, this extends the time post-thaw cells must be cultured prior to use in downstream experiments. Using a series of Design of Experiments (DOE) and mathematical modeling methods, we describe the development of a xeno-free cryomedium for use in cryopreservation of PSCs and ESCs, and a chemically defined post-thaw recovery supplement for use in recovery of PSCs, ESCs, as well as difficult-to-preserve primary cells. When used together, we show this system provides >80% direct post-thaw viability of PSCs with >70% cell survival following 24 hours post-plating. As a result of increased post-thaw survival rate, cells recover faster and are ready to passage sooner than with current solutions, while maintaining pluripotency and normal karyotype over 10 passages. Additionally, the post-thaw recovery supplement was tested in combination with other cryopreservation reagents which lead to markedly improved 24 hour post-thaw viability of difficult-to-preserve primary cells, including primary cortical neurons and human corneal epithelial cells.