Investigating solution effects injury of human T lymphocytes and its prevention during interrupted slow cooling.

Cooling rate is a critical parameter affecting the success of cell cryopreservation. Fast cooling can result in intracellular ice formation (IIF), while slow cooling can bring solution effects injury, both are detrimental to the cells. Whilst most of the studies have investigated how IIF affects cells, solution effects injury has received little attention. Here, we studied the solution effects injury of human T lymphocytes by cryomicroscopy and tested the osmoprotective ability of some frequently used cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, trehalose, urea and l-proline. We further investigated the relationship between cell volume, latent heat and solution effects cell injury. We found that solution effects injury during interrupted slow cooling was caused by high concentration of the extracellular solution rather than eutectic formation and solutes precipitation. DMSO, glycerol and trehalose can protect cells from solution effects injury, while l-proline and urea cannot under the same condition. The cell volume and latent heat are not crucial for causing solution effects injury in cells. This work confirms that high osmotic pressure, rather than eutectic formation, leads to cell injury. It also suggests that cell volume and latent heat may not be a key factor for explaining solution effects injury and its prevention in the cryopreservation of human T lymphocytes.

Cryopreservation of human T lymphocytes under fast cooling with controlled ice nucleation in cryoprotective solutions of low toxicity.

Cryopreservation of human T lymphocytes has become an essential tool for some cell-based immunotherapy. However, the cryopreservation procedure of the cells has not been systematically studied. In particular, the key factors of ice seeding and cryoprotective agents (CPA) driving the success of cryopreservation remain unclear. We systematically investigated the key factors, including cooling rate, ice-seeding temperature, CPA concentration, and types of CPA, during cryopreservation of human T lymphocytes with controlled ice nucleation. We found that ice seeding at below -10 °C could enable human T lymphocytes to be cooled at 90 °C min-1 with high relative viability and recovery after rewarming, 94.9% and 90.2%, respectively, which are significantly higher than those without ice seeding (P < 0.001). After optimization, the concentration of dimethyl sulphoxide was as low as 2% (v/v) with relative viability and recovery of 95.4% and 100.8%, respectively, at the cooling rate of 90 °C min-1 after ice seeding at -16 °C. The cryopreservation procedure developed in this study could facilitate the understanding of the mechanism for ice seeding and cell injury and offer a promising cryopreservation method with a high cooling rate and extremely low toxicity for extensive clinical application of immunotherapy.

Effect of Warming Process on the Survival of Cryopreserved Human Peripheral Blood Mononuclear Cells.

It is well known that the warming process is a critical step in cell cryopreservation, affecting the survival rate of the cryopreserved cells. However, there is a lack of understanding and optimization of the warming process for the cryopreserved human peripheral blood mononuclear cells (PBMCs) that are greatly needed for the cellular/immune therapies worldwide. In this study, the effect of the warming process on cryosurvival of the PBMCs was investigated, resulting in a recommendation of an optimal warming method. In the experiments, all PBMC samples were cooled by a fixed slow cooling process and stored in a liquid nitrogen tank. The frozen samples were then warmed in water baths with stirring at various temperatures, 37°C, 42°C, and 65°C, respectively. After thawing, PBMC's viability as well as phenotypic and functional analyses were performed and evaluated. It was shown that a relatively rapid warming process at 65°C in a water bath with stirring generated a significant improvement of cell viability, recovery, and functionality of the cryopreserved PBMCs. In addition, interferon-γ and interleukin-2 secretion were much higher in PBMCs thawed at 65°C than that in 42°C and 37°C, respectively. This study suggests that a rapid warming process at 65°C in a water bath should be used to replace the current conventional warming approach at 37°C.