Cell and Gene Therapy Catapult, 12th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK.
Asymptote, General Electric Healthcare, Sovereign House, Histon, Cambridge, CB24 9BZ, UK.
Sci Rep. 2019 Mar 4;9(1):3417. doi: 10.1038/s41598-019-39957-x.
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 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 to 113 °C min). It is only following a rapid rate of cooling (-10 °C min) that a reduction in viable cell number is observed following slow rates of warming (1.6 °C min and 6.2 °C min), but not rapid rates of warming (113 °C min and 45 °C min). 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) the ice structure appeared highly amorphous, and when subsequently thawed at slow rates (6.2 °C min 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.
对于免疫疗法的临床应用，预计细胞将被冷冻保存并运送到患者所在的地方，在那里它们将被解冻并进行给药。在细胞冷冻保存中，一个既定的观点是，在冷冻后，细胞必须迅速升温（≤5 分钟），以保持高存活率。在这项研究中，我们研究了冷却速率和升温速率对保存在传统 DMSO 基冷冻保护剂中的 T 细胞的活力和功能的相互作用，并在传统的冷冻管中进行处理。所得数据表明，只要冷却速率为-1°C min 或更慢，在检查的升温速率范围内（1.6°C min 至 113°C min），升温速率对活细胞数量没有影响。只有在快速冷却速率（-10°C min）下，才会观察到缓慢升温速率（1.6°C min 和 6.2°C min）后活细胞数量减少，但快速升温速率（113°C min 和 45°C min）则不会。低温显微镜研究表明，这种活力的丧失与升温过程中冰晶结构的变化有关。在高冷却速率（-10°C min）下，冰结构似乎高度无定形，而当随后以缓慢的速率解冻（6.2°C min 及以下）时，在解冻过程中观察到冰晶再结晶，表明冷冻细胞受到机械破坏。该数据为冷冻细胞疗法相变过程中冰晶结构依赖性行为及其对活细胞悬浮液的影响提供了一个引人入胜的见解。此外，它为 T 细胞的冷冻保存提供了一个操作范围，因为新兴行业正在为免疫疗法产品定义制剂体积和冷冻容器。
