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Primary culture and phenotyping of murine chondrocytes

Abstract

The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.

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Figure 1: Localization of articular cartilage.
Figure 2: Primary culture of immature murine articular chondrocytes.
Figure 3: Primary culture of immature murine costal chondrocytes.
Figure 4: Phenotypic characterization of primary articular chondrocytes.
Figure 5: Functional characterization of primary articular chondrocytes.

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Acknowledgements

We are grateful to Colette Salvat for her outstanding technical expertise in the development and optimization of cultured mouse articular chondrocytes and Audrey Pigenet for her high-quality technical assistance.

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Correspondence to Francis Berenbaum.

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Gosset, M., Berenbaum, F., Thirion, S. et al. Primary culture and phenotyping of murine chondrocytes. Nat Protoc 3, 1253–1260 (2008). https://doi.org/10.1038/nprot.2008.95

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