C-20: Tissue vitrification

Abstract

Application of the original tissue vitrification method developed by Fahy and used by my research group for rabbit blood vessels and articular cartilage has evolved over time. The formulation was 2.21 M propylene glycol, 3.10 M formamide and 3.10 M dimethylsulfoxide in EuroCollins solution and it was employed with multiple addition/removal steps to minimize osmotic damage to the cells. Ice-free cryopreservation using VS55, as originally described, is limited to small or thin samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides excellent extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using higher concentrations of the same cryoprotectants in EuroCollins solution has several advantages. High viability can be achieved for human and porcine articular cartilage employing an 83% formulation, VS83, in combination with multiple addition/removal steps. Lower cryoprotectant concentrations are not effective for large mammal articular cartilage. VS83 can also be used for tissue matrix preservation with retention of material properties at the expense of cell viability. Addition and removal of the cryoprotectants can be done in a single step followed by washing to minimize residual chemical concentrations. This protocol minimizes tissue cell viability and also reduces tissue immunogenicity suggesting that this vitrification strategy may be an alternative to the decellularization methods being employed for engineering tissues for medical implants. The evolution of these vitrification protocols will be reviewed drawing upon research employing natural and engineered tissues. Disclosure: Author was or is employed by Cell & Tissue Systems, Inc.