Isolation and Identification of Primary Fibroblast-like Synovial Cells from Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial hyperplasia and progressive joint destruction as its primary pathological feature. Fibroblast-like synoviocytes (FLS) are recognized as pivotal cellular components in RA pathogenesis, characterized by their aggressive, tumor-like behavior, which mediates cartilage and bone destruction. The isolation and purification of Rheumatoid arthritis Fibroblast-like synoviocytes (RA-FLS) is a critical tool for investigating disease mechanisms and evaluating potential therapeutic drugs. Here, we present a comprehensive protocol for isolating, culturing, and functionally validating primary human RA-FLS from resected synovial tissue samples obtained during knee replacement surgery in RA patients. This method utilizes fresh synovial tissue and employs a sequential enzymatic digestion process with trypsin and collagenase type II to maximize the isolation of RA-FLS from the tissue matrix. Finally, cell identification is achieved by Vimentin immunofluorescence and flow cytometry, complemented by functional assessment of TNF-α-induced proliferation. This protocol enables the acquisition of high-purity RA-FLS cultures with specific phenotypic characteristics and inflammatory responsiveness, providing researchers with a reliable method to obtain short-term cultured, patient-derived RA-FLS suitable for drug screening, mechanistic studies, and tissue engineering applications.