Optimizing CO2 normalizes pH and enhances chondrocyte viability during cold storage

Abstract

Fresh osteochondral allografts are an important treatment option for the repair of full-thickness articular cartilage defects. Viable chondrocytes within the transplanted tissue are considered important to maintaining matrix integrity. The purpose of this study is to determine whether an increase in pH decreases chondrocyte viability during cold storage and whether equilibration of Dulbecco's modified Eagle's medium (DMEM) in 5% CO(2) normalizes pH and increases chondrocyte survival during storage at 4 degrees C. Freshly isolated bovine articular chondrocytes cultured in alginate beads were stored for up to 5 days at 4 degrees C or 37 degrees C in DMEM exposed to ambient air or in DMEM equilibrated with 5% CO(2). Chondrocyte viability was determined by flow cytometry. Physiologic pH was maintained when DMEM was equilibrated with 5% CO(2), while pH increased in ambient air. After 5 days of storage at 4 degrees C, chondrocyte necrosis was higher when stored in ambient air than if equilibrated with 5% CO(2). No decrease in chondrocyte viability was observed with storage at 37 degrees C. In addition, chondrocyte viability in bovine cartilage osteochondral cores was examined after storage for 14 days at 4 degrees C in DMEM with and without HEPES, and with and without 5% CO(2). Under these conditions, the superficial layer of chondrocytes was more viable when stored in DMEM with HEPES or DMEM equilibrated with 5% CO(2) than when stored in DMEM in ambient air. This data shows that an increase in pH decreased bovine chondrocyte viability when refrigerated at 4 degrees C in DMEM, and that optimization of CO(2) normalized pH and improved chondrocyte viability during cold storage in DMEM.