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

Abstract

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.

Cryopreservation of midbrain dopaminergic neural cells differentiated from human embryonic stem cells

“…Such rapid rates of cooling result in only partial ice crystal formation, and rapid thawing some damage due to re-crystallization on warming. If cells are cryopreserved slowly in optimal conditions, then the warming rate had little impact of viability as ice crystals fully develop during the cooling [38]. In agreement with this data, we did not observe a significant improvement in cell viability for cells thawing quickly in a 37°C water-bath compared to cells thawed slowly in air at 4°C.…”

“…Cryopreserved cells are typically thawed quickly in a 37°C water-bath, and slow thawing is usually considered undesirable, which is supported by limited experimental data from the 1970s [36,37]. However, warming rates for cryopreserved mammalian somatic cells was recently revisited, and no impact of thawing conditions could be identified for cells cooled slowly at a rate of 1°C/min [38]. Only when cells were frozen quickly, 10°C/min or higher, was rapid thawing beneficial.…”

An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses

“…Most of the methods included fixation buffers, which resulted in poor lymphocyte viability (60-70%) (10) and with up to 50% T cell loss (11). The improved viability we observed in our study is likely explained by the optimized freezing media we used (12). Similar to two recent studies reporting new whole blood freezing methods (13,14), our method also (14).…”

Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution—An Optimization and Comparative Study

“…Glass transition temperature, a second-order phase transition, was determined by plotting the first derivative of heat flow over time against temperature. Two local maxima were typically found of each solution representing the glass transition and softening temperatures (Baboo et al, 2019), respectively in ascending order.…”