Slow Cooling and Controlled Ice Nucleation Enabling the Cryopreservation of Human T Lymphocytes with Low-Concentration Extracellular Trehalose

Cryopreservation of human T lymphocytes has become a key strategy for supporting cell-based immunotherapy. However, the effects of ice seeding on the cryopreservation of cells under relatively slow cooling have not been well researched. The cryopreservation strategy with a nontoxic, single-ingredient, and injectable cryoprotective solution remains to be developed. We conducted ice seeding for the cells in a solution of normal saline with 1% (v/v) dimethyl sulfoxide (Me2SO), 0.1 M trehalose, and 4% (w/v) human serum albumin (HSA) under different slow cooling rates. With the positive results, we further applied seeding in the solution of 0.2 M trehalose and 4% (w/v) HSA under the same cooling rates. The optimal concentration of trehalose in the Me2SO-free solutions was then investigated under the optimized cooling rate with seeding, with control groups without seeding, and in a freezing container. In vitro toxicity of the cryoprotective solutions to the cells was also tested. We found that the relative viability of cells (1% [v/v] Me2SO, 0.1 M trehalose and 4% [w/v] HSA) was improved significantly from 88.6% to 94.1% with ice seeding, compared with that without seeding (p < 0.05). The relative viability of cells (0.2 M trehalose and 4% [w/v] HSA) with seeding was significantly higher than that without seeding, 96.3% and 92.0%, respectively (p < 0.05). With no significant difference in relative viability between the solutions of 0.2 M trehalose or 0.3 M trehalose with 4% (w/v) HSA (92.4% and 94.6%, respectively, p > 0.05), the solution of 0.2 M trehalose and 4% (w/v) HSA was selected as the optimized Me2SO-free solution. This strategy could cryopreserve human T lymphocytes without any toxic cryoprotectant and boost the application of cell products in humans by intravenous injection, with the osmolality of the low-concentration cryoprotective solution close to that of human plasma.