Impact of cryopreservation on immune cell metabolism as measured by SCENITH.

Abstract

The dynamic functioning of immune cells is regulated by cellular metabolic processes, and there is growing interest in the study of immunometabolic correlates of dysfunctional immune responses. SCENITH is a novel flow cytometry-based technique that allows for ex vivo metabolic profiling of immune cells within heterogeneous samples. Cryopreservation of clinical samples is frequently undertaken to facilitate high throughput processing, but is thought to lead to cellular metabolic dysfunction. We aimed to investigate the impact of cryopreservation on immune cell metabolism, harnessing SCENITHs unique ability to describe the divergent bioenergetic characteristics of distinct immune cell subsets. We demonstrate that T cells undergoing activation with a CD3/CD28 stimulus are less readily metabolically reprogrammed following cryopreservation. Additionally, we find that cryopreservation introduces a time-dependent metabolic artefact that favours glycolysis and impairs oxidative phosphorylation, suggesting that cryopreservation results in mitochondrial dysfunction. Despite this artefact, SCENITH was still able to reveal the distinct bioenergetic profiles of contrasting immune cells populations following cryopreservation - for example, non-classical monocytes have a higher mitochondrial dependence than classical monocytes, and T cell CD69 expression is associated with an upregulation of glycolytic capacity. Whilst we believe that SCENITH can provide valuable information about immune cell metabolism even in cryopreserved samples, our findings have important implications for the design of future studies. Investigators should carefully consider how to process and store clinical samples to ensure that cryopreservation does not confound analyses, particularly where longitudinal sampling is required.