Chondrocyte Culture and Assay

Jeffrey Liebman1, Ronald L. Goldberg1

1 Novartis Institute of Biomedical Research, Summit, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.2
DOI:  10.1002/0471141755.ph1202s12
Online Posting Date:  May, 2001
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Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage. Cellular, biochemical and pharmacological studies of arthritis and other cartilage disorders have increasingly focused on chondrocyte function. Three methods are presented in this unit for culturing chondrocytes, and two assays are described that characterize proteoglycan synthesis, a key measure of chondrocyte function.Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage

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Table of Contents

  • Chondrocyte Culture and Assay
  • Basic Protocol 1: Chondrocyte Monolayer Culture
  • Alternate Protocol 1: Chondrocyte Culture in Alginate
  • Alternate Protocol 2: Cartilage Chip Culture
  • Support Protocol 1: Dissection of Bovine Articular Cartilage
  • Basic Protocol 2: Incorporation of 35S into Chondrocytes or Cartilage
  • Basic Protocol 3: Dimethylmethylene Blue Staining to Determine Proteoglycan Content
  • Support Protocol 2: Measurement of DNA Content
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Chondrocyte Monolayer Culture

  • Pronase E, type XIV (Sigma)
  • DMEM/antibiotic‐antifungal solution with FBS as indicated (see recipe)
  • Cartilage shavings (see protocol 4)
  • 150 to 1250 U/mg high clostripain bacterial collagenase (Worthington Biochemical)
  • 10 mg/ml ascorbic acid, sterile (optional)
  • 0.8‐, 0.45‐, and 0.22‐µm disposable filter units
  • 150‐mm sterile, plastic petri dishes
  • Sterile disposable scalpels, no. 21, no. 15, and no. 10
  • 100‐ml spinner flask with side arms
  • Impeller assembly for spinner flasks, stainless steel (Bellco)
  • Magnetic stirrer
  • 20‐µm nylon filter membrane (Spectra‐Mesh, Spectrum Laboratories)
  • Autoclaved glass funnel
  • 50‐ml centrifuge tubes, sterile
  • Clinical centrifuge
  • Culture vessels (see Table 12.2.1)
    Table 2.2.1   MaterialsRecommended Chondrocyte Monolayer Seeding Densities

    Vessel Vol. medium (ml) Cells (× 106)
    96‐well plate 0.1 0.05
    24‐well plate 0.5 0.2
    6‐well plate 2.4 1
    25‐cm2 flask 6 2.5
    100‐mm petri dish 14 5.7
    75‐cm2 flask 19 7.5

Alternate Protocol 1: Chondrocyte Culture in Alginate

  • 1.2% (w/v) alginate, sodium salt, medium viscosity (Sigma) in 0.9% sodium chloride solution (sterilize by autoclaving)
  • 102 mM calcium chloride
  • 0.9% saline
  • DMEM/10% FBS/antibiotic‐antifungal solution (see recipe for solution with 5% FBS) containing 10 to 50 µg ascorbic acid/ml
  • 20‐G needle and syringe
  • 6‐well culture dishes

Alternate Protocol 2: Cartilage Chip Culture

  • Sterile forceps

Support Protocol 1: Dissection of Bovine Articular Cartilage

  • Calf hooves (from local slaughterhouse)
  • 70% ethanol
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • DMEM/antibiotic‐antifungal solution (without serum; see recipe)
  • DMEM/10% FBS/antibiotic‐antifungal solution (see recipe)
  • Sterile disposable scalpels, no. 10, no. 15, and no. 21
  • 150‐mm petri culture dishes

Basic Protocol 2: Incorporation of 35S into Chondrocytes or Cartilage

  • ∼1000 to 1500 Ci/mmol 35S radionuclide (5 mCi/ml) in aqueous solution (NEN)
  • Chondrocyte sample: 6‐ or 24‐well dish of cultured chondrocytes (see protocol 1), alginate‐embedded chondrocytes, or cartilage chips
  • PBS, calcium‐free and magnesium‐free
  • 0.25% trypsin, without calcium or magnesium
  • DMEM/10% FBS/antibiotic‐antifungal solution (see recipe for 5% FBS solution)
  • 10× pronase E solution: 100 mg of pronase E (type XIV; Sigma; alternatively designated as “protease”) in 10 ml water (for alginate‐embedded chondrocytes only)
  • HPLC‐grade or molecular biology‐grade water
  • Scintillation cocktail
  • 0.9% saline
  • Alginate dissolving buffer (see recipe)
  • DMEM/antibiotic‐antifungal solution (see recipe)
  • 100% ethanol
  • 15‐ml polypropylene centrifuge tubes
  • 56°C incubator (nonhumidified)
  • Sephadex G‐25 molecular sieve columns (PD‐10, Pharmacia; for monolayered and alginate‐embedded chondrocytes)
  • 96‐well scintillation counting plate
  • Scintillation counter with plate reader
  • Rocking platform at 4°C
  • 1.5‐ml microcentrifuge tubes
CAUTION: Radioactive materials require special handling; radioactive waste must be disposed of appropriately.

Basic Protocol 3: Dimethylmethylene Blue Staining to Determine Proteoglycan Content

  • Medium and protease digests from cartilage or chondrocyte cultures
  • PBS‐BSA: 1× phosphate‐buffered saline (see recipe) containing 1% (w/v) purified bovine serum albumin
  • 1 mg/ml chondroitin sulfate (CS) from shark cartilage (Sigma) with 0.005% sodium azide as a preservative
  • 2× 1,9‐dimethylmethylene blue (DMB) working solution (see recipe)
  • 96‐well plates, flat clear bottom polystyrene, nonsterile (e.g., Costar)
  • Plate reader able to read absorbance between 520 to 530 nm (if this bandwidth is not available, the bandwidth 590 to 600 nm can be used).

Support Protocol 2: Measurement of DNA Content

  • Hoechst dye working solution (see recipe), prepare fresh daily
  • Lysed or protease‐digested chondrocyte samples (e.g., see protocol 1, protocol 2, or protocol 3)
  • 100 µg/ml bovine (calf) thymus DNA or other reference DNA sample (see recipe)
  • TNE buffer (see recipe)
  • 96‐well black cluster plate, clear bottom
  • Fluorescence plate reader
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Literature Cited

   Beekman, B., Verzijl, N., Bank, R.A., von der Mark, K., and TeKoppele, J.M. 1997. Synthesis of collagen by bovine chondrocytes cultured in alginate: Posttranslational modifications and cell‐matrix interaction. Exp. Cell Res. 237:135‐141.
   Goldberg, R.L., Spirito, S., Doughty, J.R., and DiPasquale, G. 1993. Release of cell surface proteoglycan from chondrocytes by interleukin‐1. Agents Actions 39:C163‐C165.
   Gregory, K.E., Marsden, M.E., Anderson‐MacKenzie, J., Bard, J.B., Bruckner, P., Farjanel, J., Robins, S.P., and Hulmes, D.J. 1999. Abnormal collagen assembly, though normal phenotype, in alginate bead cultures of chick embryo chondrocytes. Exp. Cell Res. 246:98‐107.
   Guo, J., Jourdian, G.W., and MacCallum, D.K. 1989. Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res. 19:277‐297.
   Hascall, V.C., Handley, C.J., McQuillan, D.J., Hascall, G.K., Robinson, H.C., and Lowther, D.A. 1983. The effect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture. Arch. Biochem. Biophys. 224:206‐223.
   Hauselmann, H.J., Fernandes, R.J., Block, J.A., Schmid, T.M., and Thonar, E.J.‐M.A. 1993. Adult articular chondrocytes retain their phenotype after 8 months of culture in alginate. Transactions of the Orthopedic Research Society 39th Annual Meeting, San Francisco, p. 624.
   Hauselmann, H.J., Oppliger, L., Michel, B.A., Stefanovic‐Racic, M., and Evans, C. H. 1994. Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture. FEBS Lett. 352:361‐364.
   Liu, H., Lee, Y.‐W., and Dean, M.F. 1998. Re‐expression of differentiated proteoglycan phenotype by dedifferentiated human chondrocytes during culture in alginate beads. Biochim. Biophys Acta. 1425:505‐515.
   McKenna, L.A., Gehrsitz, A., Soder, S., Eger, W., Kirchner, T., and Aigner, T. 2000. Effective isolation of high‐quality total RNA from human adult articular cartilage. Anal. Biochem. 286:80‐85.
   Morales, T.I. and Roberts, A. B. 1988. Transforming growth factor beta regulates the metabolism of proteoglycans in bovine cartilage organ cultures. J. Biol. Chem. 263:12828‐12831.
   Taskiran, D., Stefanovic‐Racic, M., Georgescu, H., and Evans, C. 1994. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin‐1. Biochem. Biophys. Res. Commun. 200:142‐148.
   van der Kraan, P., Vitters, E., and van den Berg, W. 1992. Differential effect of transforming growth factor beta on freshly isolated and cultured chondrocytes. J. Rheumatol. 19:140‐145.
   von der Mark, K. 1986. Differentiation, modulation and dedifferentiation of chondrocytes. Rheumatology 10:272‐315.
   van Osch, G.J.V.M., Van der Veen, S.W., Buma, P., and Verwoerd‐Verhoef, H.L. 1998. Effect of transforming growth factor‐beta on proteoglycan synthesis by chondrocytes in relation to differentiation stage and the presence of pericellular matrix. Matrix Biol. 17:413‐424.
   Yaron, M., Shirazi, I., and Yaron, I. 1999. Anti‐interleukin‐1 effects of diacerein and rhein in human osteoarthritic synovial tissue and cartilage cultures. Osteoarthritis Cartilage 7:272‐280.
Key References
   Goldberg, R.L. and Kolibas, L.M. 1990. An improved method for determining proteoglycans synthesized by chondrocytes in culture. Connect Tissue Res. 24:265‐275.
  This paper gives more details of the DMB assay method that is presented.
   Hauselmann, H.J., Aydelotte, M.B., Schumacker, B.L., Kuettner, K.E., Gitelis, S.H. and Thonar, E.J.‐M.A. 1992. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 12:116‐129.
  Basic characteristics of the alginate culture system are described in detail in this early paper.
   Kuettner, K.E., Pauli, B.U., Gall, G., Memoli, V.A., and Shenk, R.K. 1982. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. Isolation, culture characteristics and morphology. J. Cell Biol. 93:743‐750.
  Chondrocyte cell dissociation from cartilage and subsequent culturing is described in detail.
   Morales, T.I., Wahl, L.M., and Hascall, V.C. 1984. The effect of bacterial lipopolysaccharides on the biosynthesis and release of proteoglycans from calf articular cartilage cultures. J. Biol. Chem. 259:6720‐6729.
  Cartilage chip cultures and the [35S] assay are described.
Internet Resources
  Office of Health and Safety, Center for Disease Control, BMBL APPENDIX H. 1999. Working with Human and Other Primate Cells.
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