Cryopreserved Peripheral Blood Mononuclear Cells Recover Mitochondrial Function Following Overnight Culture Revealed by High-Resolution Respirometry

Abstract Background: High resolution respirometry of peripheral blood mononuclear cells (PBMCs) is widely used to assess mitochondrial function, but reliance on freshly isolated cells limits flexibility and sample batching. The impact of cryopreservation and post-thaw handling on PBMC mitochondrial respiration remains incompletely defined. Methods: PBMCs from healthy young adults were analysed under three conditions: freshly isolated, cryopreserved and directly thawed, and cryopreserved followed by overnight recovery in supplemented culture medium. Mitochondrial respiration was assessed using an Oroboros O2k high-resolution respirometer following plasma membrane permeabilisation and a substrate uncoupler inhibitor titration protocol. Respiration parameters included leak, oxidative phosphorylation (OXPHOS), maximal uncoupled respiration, and non-mitochondrial oxygen consumption. Results: Directly thawed PBMCs exhibited reduced ATP-coupled and maximal respiratory flux compared with fresh cells, indicating acute cryopreservation-induced mitochondrial suppression. In contrast, PBMCs undergoing overnight recovery showed substantial restoration of mitochondrial function, with OXPHOS and uncoupled respiration closely approximating those of fresh cells. Cell viability was comparable across conditions. Conclusion: Cryopreserved PBMCs can be reliably used for mitochondrial respirometry when an overnight recovery step is included. This approach enables sample storage, batching, and broader application in large-scale and retrospective human studies.