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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References
Aydelotte, M.B. & Kuettner, K.E. Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect. Tissue Res. 18, 205–222 (1988).
Kergosien, N., Sautier, J. & Forest, N. Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system. Calcif. Tissue Int. 62, 114–121 (1998).
Hauselmann, H.J. et al. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 12, 116–129 (1992).
Goldring, M.B. The role of the chondrocyte in osteoarthritis. Arthritis Rheum 43, 1916–1926 (2000).
Goldring, M.B. & Berenbaum, F. Human chondrocyte culture models for studying cyclooxygenase expression and prostaglandin regulation of collagen gene expression. Osteoarthr. Cartil. 7, 386–388 (1999).
Thirion, S. & Berenbaum, F. Culture and phenotyping of chondrocytes in primary culture. Methods Mol. Med. 100, 1–14 (2004).
Stokes, D.G., Liu, G., Dharmavaram, R., Hawkins, D., Piera-Velazquez, S. & Jimenez, S.A. Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors. Biochem. J. 360, 461–470 (2001).
Takigawa, M., Pan, H.O., Kinoshita, A., Tajima, K. & Takano, Y. Establishment from a human chondrosarcoma of a new immortal cell line with high tumorigenicity in vivo, which is able to form proteoglycan-rich cartilage-like nodules and to respond to insulin in vitro. Int. J. Cancer 48, 717–725 (1991).
Mallein-Gerin, F. & Olsen, B.R. Expression of simian virus 40 large T (tumor) oncogene in mouse chondrocytes induces cell proliferation without loss of the differentiated phenotype. Proc. Natl. Acad. Sci. USA 90, 3289–3293 (1993).
Finger, F. et al. Molecular phenotyping of human chondrocytecell lines T/C-28a2, T/C-28a4, and C-28/I2. Arthritis Rheum. 48, 3395–3403 (2003).
Loeser, R.F., Sadiev, S., Tan, L. & Goldring, M.B. Integrin expression by primary and immortalized human chondrocytes: evidence of a differential role for alpha1beta1 and alpha2beta1 integrins in mediating chondrocyte adhesion to types II and VI collagen. Osteoarthr. Cartil. 8, 96–105 (2000).
Stokes, D.G. et al. Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum. 46, 404–419 (2002).
Gibson, G.J., Schor, S.L. & Grant, M.E. Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels. J. Cell Biol. 93, 767–774 (1982).
Watt, F.M. Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture. J. Cell Sci. 89, 373–378 (1988).
Goldring, M.B., Birkhead, J., Sandell, L.J., Kimura, T. & Krane, S.M. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J. Clin. Invest. 82, 2026–2037 (1988).
Goldring, M.B. et al. Interleukin-1 betamodulated gene expression in immortalized human chondrocytes. J. Clin. Invest. 94, 2307–2316 (1994).
Demoor-Fossard, M., Redini, F., Boittin, M. & Pujol, J.P. Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation. Biochim. Biophys. Acta 1398, 179–191 (1998).
Salvat, C., Pigenet, A., Humbert, L., Berenbaum, F. & Thirion, S. Immature murine articular chondrocytes in primary culture: a new tool for investigating cartilage. Osteoarthr. Cartil. 13, 243–249 (2005).
Gabay, O. et al. Stress induced signalling pathways in hyalin chondrocytes: inhibition by Acocado Soybean Unsaponifiable (ASU). Osteoarthr. Cartil. 16, 373–384 (2008).
Gosset, M. et al. Crucial role of Visfatin in matrix degradation and PGE2 synthesis in chondrocytes: possible role in osteoarthritis process. Arthritis Rheum. 58, 1399–1409 (2008).
Lefebvre, V. et al. Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice. Matrix Biol. 14, 329–335 (1994).
Murakami, S., Kan, M., McKeehan, W.L. & de Crombrugghe, B. Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. USA 97, 1113–1118 (2000).
Murakami, S., Lefebvre, V. & de Crombrugghe, B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J. Biol. Chem. 275, 3687–3692 (2000).
Jacques, C., Recklies, A.R., Levy, A. & Berenbaum, F. HC-gp39 contributes to chondrocyte differentiation by inducing SOX9 and type II collagen expressions. Osteoarthr. Cartil. 15, 138–146 (2007).
Häuselmann, H.J. et al. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J. Cell Sci. 107, 17–27 (1994).
Reginato, A.M., Iozzo, R.V. & Jimenez, S.A. Formation of nodular structures resembling mature articular cartilage in long-term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate. Arthritis Rheum. 37, 1338–1349 (1994).
de Crombrugghe, B. et al. Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol. 19, 389–394 (2000).
Thomas, B. et al. Differentiation regulates interleukin-1beta-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase. Biochem. J. 362, 367–373 (2002).
Lemare, F., Steimberg, N., Le Griel, C., Demignot, S. & Adolphe, M. Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic responses to interleukin-1beta. J. Cell. Physiol. 176, 303–313 (1998).
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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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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DOI: https://doi.org/10.1038/nprot.2008.95
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