Background: Circulating immune cells are considered a source for biomarkers in health and disease, since they are exposed to nutritional, metabolic and immunological stimuli in the vasculature. Cryopreservation of leukocytes is routinely used for long term storage and determination of phenotypic/functional changes at a later date. Exploring the role of bioenergetics and mitochondrial (dys)function in leukocytes is often examined by using freshly isolated cells. The aim of the pilot study described herein was to assess leukocyte bioenergetics in cryopreserved cells. Methods: Leukocytes were isolated from whole blood, counted and frozen in liquid nitrogen (LN2) for a period of 3 months. Cells were thawed at regular intervals and bioenergetic analysis performed using the Seahorse XFe96 flux analyser. Results: Cryogenic storage reduced cell viability by 20%, but cell bioenergetic responses were largely intact up to 1 month storage in LN2. However, after 1 month storage, mitochondrial function was impaired as reflected by decreasing basal respiration, ATP production, MAX respiration, reserve capacity and coupling efficiency. Conversely, glycolytic activity was increased after 1 month, most notably the enhanced glycolytic response to 25mM glucose without any change in glycolytic capacity. Finally, calculation of Bioenergetic Health Index (BHI) demonstrated that this potential diagnostic parameter was sensitive to cryopreservation. Conclusion: This study has demonstrated for the first time that cryopreservation of primary immune cells modified their metabolism in a time-dependent fashion, indicated by attenuated aerobic respiration and enhanced glycolytic activity. Taken together, we recommend caution in the interpretation of bioenergetic responses or BHI in cryopreserved samples.
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