Aline Linder ¹ Kevin Portmann ¹ Klaus Eyer ^1,2*

• ¹ Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Zürich, Switzerland
• ² Department of Biomedicine, Faculty of Health, Aarhus University, Aarhus, Denmark

Human peripheral blood mononuclear cells (hPBMCs) are widely used in fundamental research and clinical applications as studying their responses to in vitro activation is an effective way to uncover functional alterations and disease-associated phenotypes. However, the availability of samples in large numbers at a specific time and location remains challenging, hence they often might preferably be collected and cryopreserved for later analysis. While the effect of cryopreservation on viability and cell surface expression is well established, changes in activity and cytokine secretion still lead to conflicting results as it is often measured in bulk or within the cells. Here, we used our platform for dynamic single-cell multiplexed cytokine secretion measurement and compared it to a traditional intracellular cytokine staining to quantify the effect of cryopreservation on cytokine secretion and expression of individual hPBMCs. Following stimulation with LPS or anti-CD3/CD28 antibodies for up to 36 or 72 h incubation, we observed distinct alterations in cytokine responses due to cryopreservation when comparing to fresh samples, but also remarkable consistencies for some cytokines and parameters. In short, the frequencies of cytokine-secreting cells in cryopreserved samples were lower for IL-6 (LPS), IL-1β (CD3/CD28) and IFN-γ (CD3/CD28), while the frequency and dynamics of IL-8 secretion were strongly impacted in all cases. We observed a large disconnect between cytokine expression and secretion for TNF-α, where the expression dramatically increased after cryopreservation, but actual secretion was, in comparison, remarkably stable. The polyfunctionality of single cells was altered by cryopreservation in specific co-secreting populations led by the effects on IL-6 or IL-8 secretion. Among immune cells, cryopreservation seemed to affect lymphocytes and monocytes differently as effects appeared early on in lymphocytes while generally observed in later time points in monocytes. Together, this study offers an in-depth quantitative insight into the biological behavior of immune cells in response to cryopreservation and stimulation, further providing some insights into conflicting results in the literature as well as guidelines for researchers planning to assess cytokine-secreting from frozen hPBMCs in immunological research or clinical 41 applications. 42