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Isolation and Culture of Murine Primary Chondrocytes

  • Anthony J. Mirando
  • Yufeng Dong
  • Jinsil Kim
  • Matthew J. Hilton
Part of the Methods in Molecular Biology book series (MIMB, volume 1130)

Abstract

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.

Keywords

Cartilage Chondrocyte Hypertrophy Mineralization Cell culture 

Notes

Acknowledgments

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.

References

  1. 1.
    Lefebvre V, Smits P (2005) Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today 75(3):200–212CrossRefPubMedGoogle Scholar
  2. 2.
    Denker AE, Haas AR, Nicoll SB, Tuan RS (1999) Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures. Differentiation 64(2):67–76CrossRefPubMedGoogle Scholar
  3. 3.
    Shukunami C, Ohta Y, Sakuda M, Hiraki Y (1998) Sequential progression of the differentiation program by bone morphogenetic protein-2 in chondrogenic cell line ATDC5. Exp Cell Res 241(1):1–11CrossRefPubMedGoogle Scholar
  4. 4.
    Aulthouse AL, Beck M, Griffey E, Sanford J, Arden K, Machado MA et al (1989) Expression of the human chondrocyte phenotype in vitro. In Vitro Cell Dev Biol 25(7):659–668CrossRefPubMedGoogle Scholar
  5. 5.
    Bjornsson S, Heinegard D (1981) Isolation and culture techniques of foetal calf chondrocytes. Biochem J 198(1):141–148CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gartland A, Mechler J, Mason-Savas A, MacKay CA, Mailhot G, Marks SC Jr et al (2005) In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method. Bone 37(4):530–544CrossRefPubMedGoogle Scholar
  7. 7.
    Glade MJ, Kanwar YS, Hefley TJ (1991) Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture. J Bone Miner Res 6(3):217–226CrossRefPubMedGoogle Scholar
  8. 8.
    Otero M, Favero M, Dragomir C, Hachem KE, Hashimoto K, Plumb DA et al (2012) Human chondrocyte cultures as models of cartilage-specific gene regulation. Methods Mol Biol 806:301–336CrossRefPubMedGoogle Scholar
  9. 9.
    von der Mark K, Gauss V, von der Mark H, Muller P (1977) Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267(5611):531–532CrossRefPubMedGoogle Scholar
  10. 10.
    Bonen DK, Schmid TM (1991) Elevated extracellular calcium concentrations induce type X collagen synthesis in chondrocyte cultures. J Cell Biol 115(4):1171–1178CrossRefPubMedGoogle Scholar
  11. 11.
    Castagnola P, Moro G, Descalzi-Cancedda F, Cancedda R (1986) Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes. J Cell Biol 102(6):2310–2317CrossRefPubMedGoogle Scholar
  12. 12.
    Grimsrud CD, Romano PR, D’Souza M, Puzas JE, Schwarz EM, Reynolds PR et al (2001) BMP signaling stimulates chondrocyte maturation and the expression of Indian hedgehog. J Orthop Res 19(1):18–25CrossRefPubMedGoogle Scholar
  13. 13.
    Leboy PS, Vaias L, Uschmann B, Golub E, Adams SL, Pacifici M (1989) Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes. J Biol Chem 264(29):17281–17286PubMedGoogle Scholar
  14. 14.
    Venezian R, Shenker BJ, Datar S, Leboy PS (1998) Modulation of chondrocyte proliferation by ascorbic acid and BMP-2. J Cell Physiol 174(3):331–341CrossRefPubMedGoogle Scholar
  15. 15.
    Zuscik MJ, Baden JF, Wu Q, Sheu TJ, Schwarz EM, Drissi H et al (2004) 5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes. J Cell Biochem 92(2):316–331CrossRefPubMedGoogle Scholar
  16. 16.
    Li TF, Chen D, Wu Q, Chen M, Sheu TJ, Schwarz EM et al (2006) Transforming growth factor-beta stimulates cyclin D1 expression through activation of beta-catenin signaling in chondrocytes. J Biol Chem 281(30):21296–21304CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gosset M, Berenbaum F, Thirion S, Jacques C (2008) Primary culture and phenotyping of murine chondrocytes. Nat Protoc 3(8):1253–1260CrossRefPubMedGoogle Scholar
  18. 18.
    Lefebvre V, Garofalo S, Zhou G, Metsaranta M, Vuorio E, De Crombrugghe B (1994) Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice. Matrix Biol 14(4):329–335CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Anthony J. Mirando
    • 1
  • Yufeng Dong
    • 1
  • Jinsil Kim
    • 1
  • Matthew J. Hilton
    • 1
  1. 1.University of Rochester Medical CenterRochesterUSA

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