Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function

Abstract: The use of cryopreserved peripheral blood mononuclear cells is common in biologicalresearch. It is widely accepted that primary cells are rendered unusable by several freezing cycles,although this practice might be very helpful when the biological material is valuable and its recollectionis impractical. To determine the extent to which primary cells undergoing repeated freezingcycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability,their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in tenhuman peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected,we observed a progressive increase in cell death percentages on three rounds of thawing, but thefrequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, weobserved a significant reduction of B cell frequency in frozen samples compared to fresh ones. Onrepeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantlydecreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.