Optimization of primary culture, inflammatory induction, and molecular analysis of fibroblast-like synoviocytes in a rat model of osteoarthritis pain.

Synovitis, driven by fibroblast-like synoviocytes (FLS), is a key early event in knee osteoarthritis (KOA). The lack of standardized protocols for FLS culture and molecular analysis hinders reproducible in vitro modeling of early KOA. We developed an integrated platform combining MIA-induced KOA pain modeling, standardized primary FLS culture, inflammation induction, and molecular analysis. We defined passages 3-7 (P3-P7) as the optimal functional passage window for FLS, during which FLS maintained > 98% purity and stable proliferation. LPS stimulation at 1000 ng/mL for 3 h robustly induced IL-1β and TNF-α expression at both mRNA and protein levels. Our optimized RT-qPCR protocols achieved high efficiency (> 91%) and linearity (r > 0.95). Using dexamethasone (DEX), we validated the platform by demonstrating suppression of NF-κB signaling and downstream inflammatory mediators (MMP3/13, VEGFA, NGF). This work provides a standardized system addressing major reproducibility challenges in FLS research.