Cryopreservation of primary human brain vascular pericytes in suspension

The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood-brain barrier. Among the commercial suppliers of HBVPs in suspension, only one provides information regarding the cryoprotectant and vehicle used, i.e., 5% dimethyl sulfoxide (Me₂SO) in complete medium, with no cryopreservation procedure given. Published protocols for the cryopreservation of brain vascular pericytes derived from human-induced pluripotent stem cells use traditional 10% Me₂SO. In this study, we characterized the cryobiological response of HBVPs using graded freezing, both in the absence of cryoprotectants, which identifies their susceptibility to the main types of cryoinjury, and in the presence of several different cryoprotectants with the aim of quantifying post-thaw outcome. We first validated our graded freezing technique using human cerebral microvascular endothelial cell/D3 clone (hCMEC/D3) by comparing the membrane integrity results in this study in the absence and presence of 5% Me₂SO plus 6% hydroxyethyl starch (HES) in hCMEC/D3 complete medium, with those previously published by our group. We then assessed the cryobiological response of HBVPs to controlled slow cooling at 1 °C/min in the absence and presence of various cryoprotectants, namely 5% Me₂SO, 10% Me₂SO, 5% Me₂SO plus 6% HES, or 10% glycerol, all in HBVP complete medium. We found that all cryoprotectants tested yielded immediate post-thaw membrane integrity >93%. Cryopreservation with 10% Me₂SO resulted in 95.3 ± 0.7% membrane integrity; using a lower concentration of Me₂SO (5%) alone, or in the presence of 6% HES, resulted in membrane integrities of 93.3 ± 0.8% and 94.2 ± 0.8%, respectively. For applications where avoiding Me₂SO is preferred, we showed that HBVPs can be cryopreserved in 10% glycerol (post-thaw membrane integrity of 95.7 ± 0.5%). The AlamarBlue reduction assay with 3 h incubation time indicated that HBVPs cryopreserved with either 5% Me₂SO, 10% Me₂SO, 5% Me₂SO plus 6% HES, or 10% glycerol exhibited post-thaw metabolic activities which were not statistically different from those of unfrozen controls. Our study presents various application-specific options for cryopreservation of primary HBVPs with validated post-thaw membrane integrity and metabolic activity.