Brian Schryver

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.

Improved thermal management of microtiter plates using the BioCision CoolSink

There is a tacit assumption that any object in direct contact with crushed ice must rapidly equilibrate near 0°C. Yet target temperature and well-to-well temperature consistency are difficult to obtain by placing microtiter plates directly on crushed ice. In addition, arranging plates directly on ice presents a number of physical drawbacks including plate instability, variation in the distribution of the plate contact, potential for ice contamination of the well contents, degradation of support integrity with time, and difficulty in leveling the plate. This study examines the thermal shift profile and equilibrium temperature properties of a room-temperature 96-well microtiter plate placed directly on ice or on a precision-machined thermoconductive interface. We demonstrate that the desired temperature management can easily be achieved through use of the BioCision CoolSink plate adaptor. Plates equilibrate more reproducibly, and at a temperature closer to that of the thermal sink. Performance can be further improved through the use of a gap-filling aqueous thermoconductive medium.