Quality Assessment by Flow Cytometry of Peripheral Blood Mononuclear Cells after Very-Long-Term Cryopreservation

BACKGROUND: Peripheral blood mononuclear cells (PBMC) are essential for immunological research and biomarker discovery in various diseases. They are widely used to monitor immune responses and evaluate treatment efficacy, including immunotherapies. Biobanking PBMCs enables longitudinal studies and batch analyses, but cryopreservation may affect cell viability, phenotype, and function. While most studies assess PBMC quality after ≤5 years of storage, little is known about samples preserved for >20 years, which is relevant for slow-progressing diseases. MATERIALS AND METHODS: = 6). PBMCs were isolated using Ficoll-based protocols, cryopreserved and stored in liquid nitrogen. After thawing, cell viability, recovery, and immune subset composition were assessed by multiparametric flow cytometry. Functional integrity was evaluated by baseline activation marker expression and viability after 24-hour culture. RESULTS: = 0.021), but viability remained high (≥79%). Flow cytometry revealed preserved staining quality and comparable frequencies of major immune subsets (T cells, B cells, monocytes, and dendritic cells) between VLTC and STC samples. Differences were observed in rarer populations such as intermediate and non-classical monocytes. Memory T cells were also decreased in VLTC samples. Baseline Th activation markers (IFN-γ, IL-4, and IL-13) were similar across groups. After 24 hours of culture, cell viability decreased similarly across VLTC and STC samples, but remained above 75%, thus supporting suitability for functional assays. CONCLUSION: PBMCs cryopreserved for over 20 years retain sufficient viability and immunophenotypic integrity for flow cytometry and short-term culture. Despite reduced recovery, these samples are suitable for immunological analyses, provided viability and storage duration are reported. Long-term PBMC biobanking is feasible for research on diseases with extended clinical timelines.