Advertisement

In Vitro Matrigel Fibroblast Invasion Assay

  • Tanja C. A. Tolboom
  • Tom W. J. Huizinga
Part of the Methods in Molecular Medicine book series (MIMM, volume 135)

Abstract

Rheumatoid arthritis is characterized by inflammation of the joints and degradation and invasion by fibroblast-like synoviocytes (FLS) of the cartilage. To assess the invasiveness of FLS an in vitro invasion assay was developed. In this invasion assay the FLS grow through an artificial matrix composed mainly of collagen IV. First, the walls of transwells are coated with paraffin to avoid meniscus formation. Subsequently, the bottoms of the transwells (on top of the membrane) are coated with a thin layer of matrigel. On top of this matrigel fibroblast-like synoviocytes are seeded at a density of 100,000 cells per milliliter. The cells are cultured in serum free medium in the inner compartment inside the transwell. To the outer compartment outside the transwell IMDM with 10% fetal calf serum and 10% NHS is added. The cells are incubated for 3 d at 37°C and 5% CO2. After 3 d the cells are fixed with 2% glutaraldehyde in phosphate buffered saline and stained with 1% crystal violet in water. The matrix on the inside of the transwells and the cells that have not grown through the matrix and the membrane are removed. The cells that have grown through the matrix and through the membrane under the transwell can be visualized by light microscopy and counted.

Key Words

Fibroblast-like synoviocytes rheumatoid arthritis (RA) invasion matrigel in vitro 

References

  1. 1.
    Firestein, G. S. (1996) Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors? Arthritis Rheum. 39, 1781–1790.PubMedCrossRefGoogle Scholar
  2. 2.
    Muller-Ladner, U., Kriegsmann, J., Franklin, B. N., et al. (1996) Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am. J. Pathol. 149, 1607–1615.PubMedGoogle Scholar
  3. 3.
    Repesh, L. A. (1989) A new in vitro assay for quantitating tumor cell invasion. Invasion Metastasis 9, 192–208.PubMedGoogle Scholar
  4. 4.
    Kleinman, H. K., McGarvey, M. L., Liotta, L. A., et al. (1982) Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21, 6188–6193.PubMedCrossRefGoogle Scholar
  5. 5.
    McGuire, P. G. and Seeds, N. W. (1989) The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells. J. Cell Biochem. 40, 215–227.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Tanja C. A. Tolboom
    • 1
  • Tom W. J. Huizinga
    • 2
  1. 1.Department of RheumatologyLeiden University Medical CentreLeidenThe Netherlands
  2. 2.Department of RheumatologyLeiden University Medical CentreRC LeidenThe Netherlands

Personalised recommendations