Impact of controlled ice nucleation on intracellular dehydration, ice formation and their implications on T cell freeze-thaw viability.

Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze-thaw as well as a controlled rate freezer to study bulk freeze-thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of -6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (-10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.