Culture and Phenotyping of Chondrocytes in Primary Culture

  • Sylvie Thirion
  • Francis Berenbaum
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 100)


The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. This chapter describes several protocols for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes) from various species (humans, mice, rabbits, and cattle). All these protocols involve primary cultures in order to limit dedifferentiation. This chapter also describes a new protocol for culturing mouse articular chondrocytes.

Key Words

Primary cell culture isolation cartilage articular chondrocytes costal chondrocytes human mice rabbit cattle 


  1. 1.
    Aydelotte, M. B., and Kuettner, K. E. (1988) Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production. Connect. Tissue Res. 18, 205–222CrossRefPubMedGoogle Scholar
  2. 2.
    von der Mark, K. and Conrad, G. (1979) Cartilage cell differentiation. Clin. Orthop. 139, 185–205PubMedGoogle Scholar
  3. 3.
    Stokes, D. G., Liu, G., Coimbra, I. B., Piera-Velazquez, S., Crowl, R. M., and Jimenez, S. A. (2002) Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum. 46, 404–419.CrossRefPubMedGoogle Scholar
  4. 4.
    Kergosien, N., Sautier, J., and Forest, N. (1998) Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system. Calcif. Tissue Int. 62, 114–121.CrossRefPubMedGoogle Scholar
  5. 5.
    Hauselmann, H. J., Aydelotte, M. B., Schumacher, B. L., Kuettner, K. E., Gitelis, S. H., and Thonar, E. J. (1992) Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 12, 116–129.PubMedGoogle Scholar
  6. 6.
    Karsenty, G. (2001) Chondrogenesis just ain’t what it used to be. J. Clin. Invest. 107, 405–407.CrossRefPubMedGoogle Scholar
  7. 7.
    Corvol, M. T., Dumontier, M. F., and Rappaport, R. (1975) Culture of chondrocytes from the proliferative zone of epiphyseal growth plate cartilage from prepubertal rabbits. Biomedicine 23, 103–107.PubMedGoogle Scholar
  8. 8.
    Mwale, F., Billinghurst, C., Wu, W., et al. (2000) Selective assembly and remodelling of collagens II and IX associated with expression of the chondrocyte hypertrophic phenotype. Dev. Dyn. 218, 648–662.CrossRefPubMedGoogle Scholar
  9. 9.
    Goldring, M. B. (2000) The role of the chondrocyte in osteoarthritis. Arthritis Rheum. 43, 1916–1926.CrossRefPubMedGoogle Scholar
  10. 10.
    Goldring, M. B. and Berenbaum, F. (1999) Human chondrocyte culture models for studying cyclooxygenase expression and prostaglandin regulation of collagen gene expression. Osteoarthritis Cartilage 7, 386–388.CrossRefPubMedGoogle Scholar
  11. 11.
    Watt, F. M. (1988) Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture. J. Cell. Sci. 89, 373–378.PubMedGoogle Scholar
  12. 12.
    Stokes, D. G., Liu, G., Dharmavaram, R., Hawkins, D., Piera-Velazquez, S., and Jimenez, S. A. (2001) 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.CrossRefPubMedGoogle Scholar
  13. 13.
    Goldring, M. B., Birkhead, J., Sandell, L. J., Kimura, T., and Krane, S. M. (1988) 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.CrossRefPubMedGoogle Scholar
  14. 14.
    Goldring, M. B., Birkhead, J. R., Suen, L. F., et al. (1994) Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes. J. Clin. Invest. 94, 2307–2316.CrossRefPubMedGoogle Scholar
  15. 15.
    Demoor-Fossard, M., Redini, F., Boittin, M., and Pujol, J. P. (1998) Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation. Biochim. Biophys. Acta 1398, 179–191.PubMedGoogle Scholar
  16. 16.
    Gibson, G. J., Schor, S. L., and Grant, M. E. (1982) 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.CrossRefPubMedGoogle Scholar
  17. 17.
    Takigawa, M., Pan, H. O., Kinoshita, A., Tajima, K., and Takano, Y. (1991) 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.CrossRefPubMedGoogle Scholar
  18. 18.
    Mallein-Gerin, F., and Olsen, B. R. (1993) 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.CrossRefPubMedGoogle Scholar
  19. 19.
    Robbins, J. R., Thomas, B., Tan, L., et al. (2000) Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1β. Arthritis Rheum. 43, 2189–2201.CrossRefPubMedGoogle Scholar
  20. 20.
    Adolphe, M., Froger, B., Ronot, X., Corvol, M. T., and Forest, N. (1984) Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium. Exp. Cell Res. 155, 527–536.CrossRefPubMedGoogle Scholar
  21. 22.
    Martin, I., Vunjak-Novakovic, G., Yang, J., Langer, R., and Freed, L. E. (1999) Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue. Exp. Cell Res. 253, 681–688.CrossRefPubMedGoogle Scholar
  22. 21.
    Kuettner, K. E., Memoli, V. A., Pauli, B. U., Wrobel, N. C., Thonar, E. J., and Daniel, J. C. (1982) Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype. J. Cell Biol. 93, 751–757.CrossRefPubMedGoogle Scholar
  23. 23.
    Domm, C., Schunke, M., Christesen, K., and Kurz, B. (2002) Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthritis Cartilage 10, 13–22.CrossRefPubMedGoogle Scholar
  24. 24.
    Zaucke, F., Dinser, R., Maurer, P., and Paulsson, M. (2001) Cartilage oligomeric matrix protein (COMP) and collagen IX are sensitive markers for the differentiation state of articular primary chondrocytes. Biochem. J. 358, 17–24.CrossRefPubMedGoogle Scholar
  25. 25.
    Rahfoth, B., Weisser, J., Sternkopf, F., Aigner, T., von der Mark, K., and Brauer, R. (1998) Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits. Osteoarthritis Cartilage 6, 50–65.CrossRefPubMedGoogle Scholar
  26. 26.
    Robbins, J. R. and Goldring, M. B. (1998) Preparation of immortalized human chondrocyte cell lines, in Tissue Engineering, Vol. 18 (Morgan, J. R., and Yarmush, M. L., eds.), Humana, Totowa, NJ, pp. 173–192.CrossRefGoogle Scholar
  27. 27.
    Goldring, M. B. (1996) Human chondrocyte cultures as models of cartilage-specific gene regulation, in Human Cell Culture Protocols, Vol. 2 (Gareth, E. J., ed.), Humana, Totowa, NJ, pp. 217–232.CrossRefGoogle Scholar
  28. 28.
    Aulthouse, A. L., Beck, M., Griffey, E., et al. (1989) Expression of the human chondrocyte phenotype in vitro. In Vitro Cell Dev. Biol. 25, 659–668.CrossRefPubMedGoogle Scholar
  29. 29.
    Carrascosa, A., Audi, L., and Ballabriga, A. (1985) Morphologic and metabolic development of human fetal epiphyseal chondrocytes in primary culture. Pediatr. Res. 19, 720–727.CrossRefPubMedGoogle Scholar
  30. 30.
    Reginato, A. M., Iozzo, R. V., and Jimenez, S. A. (1994) 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.CrossRefPubMedGoogle Scholar
  31. 31.
    Stove, J., Gerlach, C., Huch, K., et al. (2001) Gene expression of stromelysin and aggrecan in osteoarthritic cartilage. Pathobiology 69, 333–338.CrossRefPubMedGoogle Scholar
  32. 32.
    Kawiak, J., Moskalewski, S., and Darzynkiewicz, Z. (1965) Isolation of chondrocytes from calf cartilage. Exp. Cell Res. 39, 59–68.CrossRefPubMedGoogle Scholar
  33. 33.
    Nedelec, E., Abid, A., Cipolletta, C., et al. (2001) Stimulation of cyclooxygenase-2-activity by nitric oxide-derived species in rat chondrocyte: lack of contribution to loss of cartilage anabolism. Biochem. Pharmacol. 61, 965–978.CrossRefPubMedGoogle Scholar
  34. 34.
    Okazaki, M., Higuchi, Y., and Kitamura, H. (2003) AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes. Osteoarthritis Cartilage 11, 122–132.CrossRefPubMedGoogle Scholar
  35. 35.
    Gouze, J. N., Bordji, K., Gulberti, S., et al. (2001) Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1β-mediated effects in rat chondrocytes. Arthritis Rheum. 44, 351–360.CrossRefPubMedGoogle Scholar
  36. 36.
    Gouze, J. N., Bianchi, A., Becuwe, P., et al. (2002) Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-k B pathway. FEBS Lett. 510, 166–170.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Sylvie Thirion
    • 1
  • Francis Berenbaum
    • 2
  1. 1.UMR CNRS 6544, Laboratoire Interactions Cellulaires Neuroendocriennes, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine NordUniversité Aix-Marseille IIMarseilleFrance
  2. 2.UPRES-A CNRS 7079Université Pierre et Marie CurieParisFrance

Personalised recommendations