A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis

The cryopreservation of human lymphocytes is an essential step for the achievement of several cellular therapies. Besides, T cells are considered as promising actors in cancer therapy for their cytotoxic and regulatory properties. Consequently, the development of tools to monitor the impact of freezing and thawing processes on their fine distribution may be an asset to achieve quality control in cellular therapy. In this study, the phenotypes of freshly isolated human mononuclear cells were compared to those observed following one cycle of cryopreservation and rest periods 0 h, 1 h and 24 h after thawing but before staining. T cells were scrutinized for their distribution according to naive, memory effector, regulatory and helper subsets. Flow cytometry analyses were done using eight-color antibody panels as proposed by the Human Immunophenotyping Consortium. Data were further analyzed by using conventional directed gating and clustering software, namely SPADE and viSNE. Overall, SPADE and viSNE tools were very efficient to monitor the outcome of PBMC populations and T cell subsets. T cells were more sensitive to cryopreservation than other cells. Our results indicated that submitting the thawed cells to a 1 h rest period improved the detection of some cell markers when compared to fresh samples. In contrast, cells submitted to a 24 h rest period, or to none, were less representative of fresh sample distribution. The heterogeneity of PBMC, as well as the effects of freeze-thaw cycle on their distribution, can be easily monitored by using SPADE and viSNE.