The Impact of Varying Cooling and Thawing Rates on the Quality of Cryopreserved Human Peripheral Blood T Cells

For the clinical delivery of immunotherapies it is anticipated that cells will be cryopreserved and shipped to the patient where they will be thawed and administered. An established view in cellular cryopreservation is that following freezing, cells must be warmed rapidly (≤5 minutes) in order to maintain high viability. In this study we examine the interaction between the rate of cooling and rate of warming on the viability, and function of T cells formulated in a conventional DMSO based cryoprotectant and processed in conventional cryovials. The data obtained show that provided the cooling rate is -1 °C min^-1 or slower, there is effectively no impact of warming rate on viable cell number within the range of warming rates examined (1.6 °C min^-1 to 113 °C min^-1). It is only following a rapid rate of cooling (-10 °C min^-1) that a reduction in viable cell number is observed following slow rates of warming (1.6 °C min^-1 and 6.2 °C min^-1), but not rapid rates of warming (113 °C min^-1 and 45 °C min^-1). Cryomicroscopy studies revealed that this loss of viability is correlated with changes in the ice crystal structure during warming. At high cooling rates (-10 °C min^-1) the ice structure appeared highly amorphous, and when subsequently thawed at slow rates (6.2 °C min^-1 and below) ice recrystallization was observed during thaw suggesting mechanical disruption of the frozen cells. This data provides a fascinating insight into the crystal structure dependent behaviour during phase change of frozen cell therapies and its effect on live cell suspensions. Furthermore, it provides an operating envelope for the cryopreservation of T cells as an emerging industry defines formulation volumes and cryocontainers for immunotherapy products.