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

Jasmin Baboo,Fernanda Fonseca,Peter Kilbride,Julie Meneghel,Stuart Milne,Magdalena Blanco,G John Morris,Alex Nancekievill,Mike Delahaye,Nick Gaddum

doi:10.1038/s41598-019-39957-x

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.

Highlights

Www.nature.com/scientificreports on the outcomes of cryopreservation of T cells has been published
Whilst this may be true for spermatozoa which have been cooled rapidly with glycerol as a cryoprotectant[22,23], the evidence for somatic mammalian cells which have been cooled at slow rates with DMSO as cryoprotectant is lacking[24,25]
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

Summary

Introduction

Www.nature.com/scientificreports on the outcomes of cryopreservation of T cells has been published. Because of the need to maintain sterility of parenterally delivered treatments classical screwcap cryovials will likely be replaced with newer hermetically sealed vials which employ plastics with lower thermal conductivity and higher capacity, or with cryobags of large volumes (typically 50 mL to 250 mL) This would be expected to influence the rate of thawing of the samples. An estimation is presented to allow scaling from the conventional cryovial format used in this study In this case, predictions are made as to whether the rate of thawing would become limiting to the efficacy of the clinical treatment when using new generation vials of lower conductivity/larger capacity and bags of larger volumes. T cells were originally expanded from a fresh leukapheresis pack by stimulation with GMP grade

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