1 Cryoprotectant agent toxicity in porcine articular chondrocytes 2 3

23 24 Large articular cartilage defects have proven difficult to treat and often result in 25 osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an 26 increased supply of tissues for osteochondral allograft but cryoprotective agents are 27 required; however, few studies have been performed on the toxicity of these agents. This 28 study was designed to determine the order of toxicity of five commonly used 29 cryoprotectant agents as well as interactions that occur between them. Isolated porcine 30 articular chondrocytes were exposed to individual cryoprotectant agents and 31 combinations of these agents at 1M and 3M concentrations for 5 minutes and 120 32 minutes. Cell viability was determined using membrane integrity dyes and a metabolic 33 activity assay. Subsequently, a regression analysis based study was undertaken to extract 34 the maximum amount of information from this data. Results of this study demonstrated 35 that all 1M solutions were minimally toxic. The 3M solutions demonstrated varying 36 toxicity after 120 minutes. Ethylene glycol and glycerol were less toxic than propylene 37 glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were 38 less toxic than single cryoprotectant agents at the same concentration. This is the most 39 comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes 40 and has resulted in important information regarding the order of toxicity and interactions 41 that occur between these agents. 42