Isolation and Culture of Murine Primary Chondrocytes
To identify factors that are necessary and sufficient for chondrocyte hypertrophic differentiation and cartilage matrix mineralization, primary chondrocyte culture models have been developed. Here we describe the isolation, short-term and long-term culture, and analysis of primary costal chondrocytes from the mouse. Briefly, sternae and rib cages from neonatal pups are dissected, and chondrocytes are isolated via enzymatic digestions. Chondrocytes are then plated at high density and cultured in the presence of ascorbic acid and beta-glycerophosphate as well as various recombinant proteins to promote or inhibit hypertrophic differentiation. We also describe the use of adenoviruses to recombine floxed alleles and over-express genes within these cultures. Finally, we detail methods for alkaline phosphatase and alizarin red staining that are used to visualize chondrocyte maturation and cartilage matrix mineralization.
KeywordsCartilage Chondrocyte Hypertrophy Mineralization Cell culture
We would like to acknowledge Dr. Tian-Fang Li, Dr. Jennifer H. Jonason, and Zhaoyang Liu for their help in the initial protocol design and real-time qPCR assays. These studies were supported via the following NIH mechanisms: R01 grants (AR057022 and AR063071), R21 grant (AR059733), and P30 Core Center grant (AR061307) to M.J.H.
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