Precultivation of cells subjected to long-term cryopreservation negatively affects proliferative activity of T lymphocytes

Peripheral blood mononuclear cells are a valuable biological material for most immunological studies. Cryopreservation of peripheral blood mononuclear cells provides access to large amounts of biomaterial at any time. Since cryopreservation can affect various cell parameters, several authors have proposed introducing a precultivation step for thawed cells to restore their functions. The aim of the study is to evaluate the effect of preculturing long-term cryopreserved cells on T lymphocyte proliferative capacity. Venous blood obtained from 18 relatively healthy volunteers who signed informed consent was used. Mononuclear leukocytes were isolated by centrifugation in a diacoll density gradient using a standard method. The resulting cells were subjected to controlled freezing at -80 °C for 24 hours before being transferred to liquid nitrogen. The duration of sample storage was 40±1.4 months. After thawing, the cells were divided into two parts; one part was immediately stained with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), stimulated with phytohemagglutinin, and cultured in complete culture medium containing interleukin-2 for 7 days. The second part of the cells was precultivated in complete culture medium (18 hours) and then treated as described above. T lymphocyte proliferation was assessed by flow cytometry. It was found that the preculture stage did not affect the relative counts of proliferating T lymphocytes. However, the number of daughter generations formed by both CD4⁺ and CD8⁺T lymphocytes was reduced in samples with precultured cells. Additionally, a significant increase in the proportion of dying elements among dividing CD4⁺T cells was observed. This consequence of the preculture stage was not found in the pool of CD8⁺T lymphocytes. Thus, preculturing significantly increases the percentage of T lymphocytes dying during division. This phenomenon was only detected among CD4⁺T cells, which appears to reflect their greater sensitivity to in vitro death compared to CD8⁺T lymphocytes.