In vitro matrigel fibroblast invasion assay

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 degrees 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.