Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research

Abstract

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze–thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Measuring anti-islet autoimmunity in mouse and human by profiling peripheral blood antigen-specific CD4 T cells

“…2, freeze-thaw effects on the samples led to an almost complete disappearance of cells expressing large numbers of activation genes (cluster #1), whereas cells with low expression were mostly preserved (cluster #2). The fragility of activated T cells to cryopreservation has long been described ( 26 , 27 ) but is often overlooked or ignored because of the logistics associated to using fresh PBMCs.…”

“…From our work, one more element must be discussed in relationship to using tetramers to isolate cells: the need to use fresh blood. As shown, the freeze-thaw cycle is detrimental to the activated T cell population ( 26 , 27 ), the one with the highest value for diagnosis. This necessity prevents the use of frozen samples from biobanks.…”

Cryopreserved anti-CD22 and bispecific anti-CD19/22 CAR T cells are as effective as freshly infused cells

“… 17 Frozen CAR T cells also exhibit different gene expression, with increased expression of genes associated with apoptosis and cell cycle damage compared with fresh cells. 18 Furthermore, it is known that different lymphocyte subsets have varying tolerance to the freeze-thaw process, 19 , 20 , 21 so results from one cell type cannot necessarily be extrapolated to others. The added delay in CAR T cell infusion when cells need to be cryopreserved and shipped may also lead to disease progression and subsequently worse outcomes, but it is likely necessary to facilitate the commercial development of this novel treatment approach.…”

Monocyte and Macrophage Function Diversity

“…One of the important aspects relevant to macrophage studies addressed by Haider et al [ 13 ] is how or if different cell preservation techniques influence the quantitative and functional analyses of immune cells. The authors analyzed how the cryopreservation of mononuclear cells (MNCs), i.e., blood cells with a single, round nucleus, such as lymphocytes and monocytes, taken from 15 individuals, affected cell quantity and inflammatory responses.…”