Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting mainly the synovial joints. The chronic phase of disease is characterized by the hyperproliferation of synovial tissue and formation of pannus, resulting in the progressive erosion of cartilage and bone tissue and leading to disability. Despite the uncertainties on the autoimmune versus autoinflammatory nature of RA, the requirement of non-immune cell function such as synovial fibroblasts (SFs) in the loss of tissue integrity has been widely accepted 1. SFs are CD45-negative cells of mesenchymal origin involved in supporting and lubricating the joint by providing nutrients and proteoglycans 2. Interestingly, it was shown that human RA-SFs are activated and contribute to hyperplasia and destruction 3 and mixed populations of human synoviocytes when implanted into Severe Combined Immuno-Deficiency (SCID) mice retain their ability to destroy cartilage in the absence of a functioning immune system 4. Indeed, cells from arthritic joints in culture show increased invasiveness, matrix degradation and increased cell adhesion 5.
A murine experimental paradigm that strongly supports this notion came from the generation of human-TNF transgenic mice (henceforth noted as Tg197), a representative model of chronic inflammatory polyarthritis 6. In the Tg197 model, TNF signalling through TNFRI in SFs appears to be sufficient for the orchestration of full-blown pathology 7. Additionally, SFs from the same mouse can transfer arthritic pathology to healthy recipients upon intra-articular transfer 8. Further understanding of the biology of the SF and the mechanisms leading to its arthritogenic properties should be key to unravelling the contribution of this specific cell type in the pathogenesis of chronic arthritis in human.
A well-standardized protocol for ex vivo culturing of murine SFs is currently missing from the literature and should prove useful both for unravelling the importance of SFs in disease as well as for the development of novel therapeutic approaches for several chronic joint diseases. We present here a detailed protocol for efficient isolation of primary murine SFs, which after 20 days of cell culturing, retain their original characteristics of constitutive CD90.2, VCAM-1 and ICAM-1 expression. The method presented is time-effective, and the materials needed are minimum compared to other published methods for mouse tissue 9-11. More importantly, it results in an SF population of high yield and purity.