The effects of Cryopreservation on PBMCs transcriptome profile

Cryopreservation is a key method for long-term storage of biological specimens. The development of optimal cryopreservation condition will reduce the damage from the storage as least as possible. The effect of the cryopreservation condition we used on peripheral blood mononuclear cells (PBMCs) has been previously evaluated with microarray analysis. The emerging single-cell RNA sequencing (scRNA-seq) technology enable deeper exploring cellular heterogeneity and function. In the current study, we further optimized the cryopreservation procedure using FACS analysis, and the method were further evaluated by ScRNA-Seq for two different length of storage time: six months and 12 months in comparison to fresh cells. We identified major immune cell types from both fresh and recovered cryopreserved PBMCs, including monocytes, dendritic cells (DCs), natural Killer (NK) cells, CD4+ T cells, CD8+ T cells, and B cells. The cell viability of all identified immune cell types was relatively stable during the initial 6-month of cryopreservation, while showed10 ~ 40 % decline after cryopreserved for 12 months for different cell types. Furthermore, transcriptome profile of cryopreserved samples did not show obvious perturbation during whole 12 months testing time, although a few key genes involved in stress response or apoptosis, exhibited significant change, especially in monocytes, DC and NK cells. Our results validate that the optimized cryopreservation is a reliable procedure for maintaining these major immune cell types in PBMCs, also highlight the importance of careful assessment of individual sample as variation could be introduced by cryopreservation. Fresh and thawed PBMCs samples from three donors were analyzed by FACS, and scRNA-seq to study the impact of cryopreservation on cell viability and transcriptome profile.