Key events during the cryopreservation of t cells

Abstract

Background & Aim Slow, controlled cooling is essential for successful cryopreservation of t cells. In practice, samples are typically cooled at a controlled rate of around 1°C min−1 before being transferred at around -80°C to -100°C. However, other than dehydration, the physical and biological events occurring as the temperature is lowered in t cells (and mammalian cells more widely) have been under-studied. Placing samples in the liquid or vapor phase of nitrogen too early could result in rapid cooling rates, leading to intracellular ice formation and cell death. However, there is little basis in the literature for setting the controlled cooling endpoint at -80°C or below. The present work explored the critical physical and biological events during the cooling of t cells (immortalized jurkat t cell line) through a combination of biological and physical measurements (Differential Scanning Calorimetry and Fourier transformed Infra-Red Spectroscopy). Methods, Results & Conclusion During cooling in a VIA Freeze controlled rate freezer, cells were seen to undergo a membrane transition from a liquid crystal to a gel state at -1.0°C ± 0.8°C. Ice nucleation occurred at -7.7°C ± 1.4°C in 1mL cryovials and was characterized by a rapid release of heat and macroscopic solidification of the sample. Cells dehydrate on cooling after ice nucleation to remain in osmotic equilibrium with the extra-cellular channels between ice crystals. A further transition was observed using DSC at -46.9°C ± 1.3°C. This transition was only present in cryovials during cooling when cells were present and is an intracellular glass transition. The intracellular glass transition temperature corresponded to the cells becoming osmotically inactive, reaching their maximally dehydrated state. Biologically samples following controlled cooling and plunged into liquid nitrogen below this temperature showed good post-thaw outcome, whereas samples plunged into liquid nitrogen above this point had substantial reduction in post-thaw viability and function. An extracellular glass transition at -123°C was observed and below this temperature samples could be safely stored and shipped. These results suggest that controlling cooling until -47°C is reached in t cells is critical for a successful cryopreservation, and no biological advantage is conferred by further cooling. Shorter controlled-rate cooling cycles could thus safely be applied, saving time and potentially allowing more cryopreservation cycles to be completed a day.