Standardized cryopreservation of pluripotent stem cells.

The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled-rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled-rate freezers. The protocol also eliminates the requirement for isopropanol, avoiding the hazards, on-going cost, and inconsistencies associated with its use and disposal. It provides a clinically relevant, inexpensive, reliable, and user-friendly method that successfully prepares cells for long-term cold storage and ensures maximum levels of cell viability post thaw.