Protocol for Cryopreservation of Particulated Articular Cartilage by Vitrification

Transplantation of particulated articular cartilage (AC) is an established surgical procedure that produces promising clinical results while potentially preventing joint deterioration into osteoarthritis. This chapter documents a patented vitrification protocol that is able to cold preserve particulated AC tissue, thereby maintaining cell viability and matrix integrity. Mathematical modeling was used to develop this two-step, dual-temperature, multi-cryoprotectant loading protocol. The cryoprotectant loading consists of exposing the particulated AC to two sequential cryoprotectant cocktails at different temperatures and for different durations. The fresh particulated AC is incubated in the first cryoprotectant solution that contains 3 M dimethyl sulfoxide and 3 M ethylene glycol at 0°C for 10 min. Subsequently, the particulated AC is transferred to the second cryoprotectant solution that consists of 3 M dimethyl sulfoxide, 3 M ethylene glycol, and 2 M propylene glycol at -10°C for an additional 20 min. After cryoprotectant loading, the particulated AC is loaded in a cryovial and plunged directly into liquid nitrogen for vitrification and storage. Rewarming is performed using a 37°C water bath for approximately 30 s and the cryoprotectants are washed from the rewarmed sample using 30 mL of DMEM/F-12-HEPES for 45 min at 4°C on a shaker.