An efficient combination of a CPA loading protocol, metal container, and storage method for successful articular cartilage vitrification

Abstract

Successful cryopreservation of articular cartilage (AC) depends on a number of variables, including heat transfer, sample packaging for cryogenic storage, cryoprotectant agent (CPA) concentration (toxicity), and CPA permeation into AC. In the first experiment of the present study, we used a combination of our established vitrification protocol (430-min multi-step loading Protocol 8) and the metal Ovarian Tissue Cryosystem (OTC) as a closed vitrification-storage-rewarming container to vitrify 7-mm diameter porcine osteochondral dowels with 2 methods of storage (storage in the OTC with or without a surrounding vitrification solution). In the second experiment, in an attempt to introduce a more reproducible and safe vitrification protocol, we employed our successful Protocol 8 with the OTC as the CPA loading container and a cryobag as the storage container (with or without surrounding vitrification solution). In both experiments, post-warming normalized chondrocyte viabilities were assessed. The results of the first experiment demonstrated that a combination of the established step-wise CPA loading-vitrification-rewarming protocol (Protocol 8) with the metal OTC as the only container through all steps of CPA loading, vitrification, and rewarming produced a positive result. In the second experiment, combination of Protocol 8, the OTC as the CPA loading system and a cryobag as the storage container in both vitrified groups with or without solution resulted in high normalized chondrocyte viabilities (94.63 % and 96.17 %, respectively) post vitrification in 7-mm diameter porcine osteochondral dowels. Overall, this study demonstrated that by employing our established Protocol 8, using the metal OTC as a CPA loading container and a cryobag as the storage method we could achieve a reproducible method for clinical applications involving vitrified articular cartilage.