Establishing cryopreservation protocol for human Peripheral Blood Mononucleated Cells (PBMC) for generation of NK cells for immunotherapy

Abstract

Background & Aim The human peripheral blood mononucleated cells (PBMC) which largely consist of lymphocytes have been identified as one of the potential sources for cell-based immunotherapy due to its ease of isolation and expansion. These valuable mononucleated cells can be stored under cryopreservation state before the mononucleated cells are thawed for therapeutic purposes or expansion. This study was performed to determine the feasibility to isolate and cryopreserved PBMC with no significant effect on NK cells phenotype and proliferation ability. Methods, Results & Conclusion Method PBMC were isolated from whole blood samples collected from consenting six healthy donors using density gradient centrifugation. Cells viability and total nucleated count after isolation was determined before dividing the PBMC population into two groups. The mononucleated cells in one group were were analyzed as freshly isolated PBMC (Group A) while the mononucleated cells in the other group were cryopreserved for 1 month (Group B) under liquid nitrogen before analysis. Both groups were analysed for NK cells phenotype and ability to proliferate. Results The mean post isolated PBMC was 0.98 × 106 cells/ml whole blood. The cell viability was high at 99.2±0.75% for Group A and 92.5±2.5% for Group B. We compared the observed expression of NK cells phenotype (CD56+CD3−) for both groups and found no significant difference in expression prior to culture. NK cells in both groups also exhibit the ability to proliferate under in-vitro culture for 21 days. Upon harvest, the expanded cells consisting mainly of NK cells with a mean of 75.1±16.6% (Group A) and 69.6±30.6% (Group B) and were on average (Group A: 61.8%, Group B: 54.6%) higher than in NK cells proportion prior to culture. The mean fold expansion on NK cells were 96.1±68.2 fold (Group A) and 122.2±30.9 fold (Group B). Conclusion We conclude that it is feasible to cryopreserve PBMC for 1 month for generation of NK cells without any significant effect on its phenotype and proliferation ability. However, it is recommended to repeat this study with PBMC which have been cryopreserved for a longer period. From this study, we can conclude that it is feasible to cryopreserve PBMC for generation of NK cells for immunotherapy.