Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

Abstract

Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

Keywords: PBMC; T-cell functionality; cryopreservation; recovery; temperature fluctuations; viability.

FIG. 1.

Automated robotic system with robotic arm and sample cabinet. Robotic arm changed vertical position. Samples exposed to room temperature and temperature below −130°C. Temperature shift simulates storage conditions during biobanking. Modified from Germann et al.

FIG. 2.

Cycle program in detail. After starting the cycle program, the sample cabinet is moved into the cold gas phase above liquid nitrogen until a temperature below −130°C is reached in the reference sample. Next, the sample cabinet moves up into a room temperature zone until a defined temperature of −60°C is reached in the reference sample. This process is repeated until the last remaining samples are exposed to 350 cycles.

FIG. 3.

PBMC viability after thawing (A) and after overnight culture (B). Figures show the PBMC viability after 0–350 temperature cycles. PBMC viability determined directly after thawing and after overnight culture. Figures show mean ± standard deviation. *Statistical difference to 0 cycles with p < 0.05. PBMC, peripheral blood mononuclear cell.

FIG. 4.

PBMC recovery after thawing (A) and after overnight culture (B). Figures show the PBMC recovery after 0–350 temperature cycles. PBMC recovery determined directly after thawing and after overnight culture. Figures show mean ± standard deviation. *Statistical difference to 0 cycles with p < 0.05.

FIG. 5.

Reduction of antigen-specific T cell response. (A) Shows reduction of T cell response after stimulation with CEF peptide pool and (B) after stimulation with CMV peptide pool. To show the reduction in a clearer way, a linear fit was performed and is presented as a line. Figures show reduction of immune response after 50–350 temperature cycles in comparison with 0 cycles. Figures show mean ± standard deviation of four donors. The gradients are significantly different from zero temperature cycles with p < 0.05.