Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA.
Johnson and Johnson Innovative Medicine, Malvern, PA, 19355, USA.
Int J Pharm. 2024 Nov 15;665:124694. doi: 10.1016/j.ijpharm.2024.124694. Epub 2024 Sep 10.
Cryopreservation is important in manufacturing of cell therapy products, influencing their safety and effectiveness. During freezing and thawing, intracellular events such as dehydration and ice formation can impact cell viability. In this study, the impact of controlling the ice nucleation temperature on intracellular events and viability were investigated. A model T cell line, Jurkat cells, were evaluated in commercially relevant cryoformulations (2.5 and 5 % v/v DMSO in Plasma-Lyte A) using a cryomicroscopic setup to monitor the dynamic changes cells go through during freeze-thaw as well as a controlled rate freezer to study bulk freeze-thaw. The equilibrium freezing temperatures of the studied formulations and a DMSO/Plasma-Lyte A liquidus curve were determined using DSC. The cryomicroscopic studies revealed that an ice nucleation temperature of -6°C, close to the equilibrium freezing temperatures of cryoformulations, led to more intracellular dehydration and less intracellular ice formation during freezing compared to either a lower ice nucleation temperature (-10 °C) or uncontrolled ice nucleation. The cell membrane integrity and post thaw viability in bulk cryopreservation consistently demonstrated the advantage of the higher ice nucleation temperature, and the correlation between the cellular events and cell viability.
冷冻保存对于细胞治疗产品的制造非常重要，影响其安全性和有效性。在冷冻和解冻过程中，细胞内的事件，如脱水和冰形成，会影响细胞活力。在这项研究中，研究了控制冰核温度对细胞内事件和活力的影响。使用冷冻显微镜装置评估了商业相关的冷冻配方（Plasma-Lyte A 中的 2.5%和 5%v/v DMSO）中的模型 T 细胞系 Jurkat 细胞，以监测细胞在冷冻-解冻过程中的动态变化，以及使用控速冷冻机研究批量冷冻-解冻。使用 DSC 确定了所研究配方的平衡冷冻温度和 DMSO/Plasma-Lyte A 固相线曲线。冷冻显微镜研究表明，冰核温度为-6°C，接近冷冻配方的平衡冷冻温度，与较低的冰核温度（-10°C）或非受控冰核相比，在冷冻过程中导致更多的细胞内脱水和更少的细胞内冰形成。在批量冷冻保存中，细胞膜完整性和解冻后活力始终显示出较高冰核温度的优势，以及细胞事件和细胞活力之间的相关性。
