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Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures

Nature Protocols volume 4, pages 928–938 (2009)Cite this article

Abstract

The culture of chondrocytes embedded within agarose hydrogels maintains chondrocytic phenotype over extended periods and allows analysis of the chondrocyte response to mechanical forces. The mechanisms involved in the transduction of a mechanical stimulus to a physiological process are not completely deciphered. We present protocols to prepare and characterize constructs of murine chondrocytes and agarose (1 week pre-culture period), to analyze the effect of compression on mRNA level by RT-PCR (2–3 d), gene transcription by gene reporter assay (3 d) and phosphorylation state of signaling molecules by western blotting (3–4 d). The protocols can be carried out with a limited number of mouse embryos or newborns and this point is particularly important regarding genetically modified mice.

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Figure 1: Embedding and culture of mouse chondrocytes in agarose gel.
Figure 2: Immunohistological analysis of extracellular matrix deposition in chondrocytes cultured in agarose gels over 6 d.
Figure 3: Compression device.
Figure 5: Analysis of gene expression regulation in chondrocytes embedded in agarose gels submitted to dynamic compression.
Figure 6: Analysis of the activation of MAP (mitogen-activated protein) kinases in chondrocytes embedded in agarose gels subjected to dynamic compression.
Figure 7: Analysis of the activity of the COL2A1 gene promoter in chondrocytes embedded in agarose gels immediately after being subjected to dynamic loading at 0.2 Hz for 30 min.
Figure 4

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Acknowledgements

We thank Novotec (Lyon) for the generous gift of the anti-aggrecan antibody and the technical facilities of IFR 128 (BioSciences Gerland—Lyon Sud) for the quantitative PCR analyses. We are grateful to Karine Duroure for technical assistance. We also thank Professor Philippe Galéra (Caen University) for the gift of the p3 plasmid carrying a fragment of the human type II collagen gene promoter. This study was funded by Rhône-Alpes Region (Emergence 2005), ANR (TECSAN Promocart 2006), CNRS and the Lyon 1 University. C.B. was supported by the French Ministère de la Recherche.

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  1. UMR5086, CNRS, IFR128, IBCP (Institut de Biologie et Chimie des Protéines), Université de Lyon, Lyon, France

    Carole Bougault, Anne Paumier, Elisabeth Aubert-Foucher & Frédéric Mallein-Gerin

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Correspondence to Frédéric Mallein-Gerin.

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Bougault, C., Paumier, A., Aubert-Foucher, E. et al. Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures. Nat Protoc 4, 928–938 (2009). https://doi.org/10.1038/nprot.2009.63

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