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

Summary

This protocol describes the method for isolating and identifying high-purity primary fibroblast-like synoviocytes (FLS) from synovial tissue of rheumatoid arthritis patients for reliable disease modeling and drug screening.

Abstract

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.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by symmetric polyarthritis of peripheral joints, with an estimated global prevalence of 0.5%-1% and the potential to induce various systemic and extra-articular complications^1,2,3,4,5. The core pathological feature of RA is chronic synovitis mediated by autoimmune responses, which further leads to the formation of invasive pannus tissue that erodes articular cartilage and subchondral bone^6,

Protocol

This study collected synovial tissue from rheumatoid arthritis patients undergoing joint replacement surgery at China-Japan Friendship Hospital. All patients provided written informed consent prior to surgery. The collected information and biological samples were handled with strict measures to ensure patient privacy and data security. Any remaining synovial tissue specimens were disposed of in accordance with biosafety regulations. The use of knee joint synovial tissue from rheumatoid arthritis patients in this study was approved by the Ethics Committee of China-Japan Friendship Hospital (Approval No.: 2025-KY-223). The reagents and the equipment used are listed in t....

Results

Using the protocol, we successfully isolated short-term cultured, patient-derived RA-FLS. A schematic diagram of the procedural workflow is provided (Figure 1). Under light microscopy, the RA-FLS exhibited a predominant morphological phenotype of spindle-shaped and irregular polygonal cells. The cells displayed extensive cytoplasmic spreading with elongated processes. Cell borders appeared slightly indistinct in areas of cell-to-cell contact or overlap. The majority of cells presented a rela.......

Discussion

This protocol establishes a reproducible method for the isolation and short-term culture of RA-FLS from RA patients, with its success evidenced by high cell viability, a characteristic phenotype, specific marker expression, and functional responsiveness to inflammatory stimuli.

Critical steps and technical considerations
The reliability of this method hinges on several critical steps. First, fresh synovial tissue should be subjected to enzymatic digestion promptly within.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488-conjugated anti-rabbit IgG	Zhongshan Jingqiao Biotechnology	ZF-0511
0.25% Trypsin-EDTA (1X)	Gibco	25200-072
4% Paraformaldehyde	Solarbio	P1110
Alexa Flour674 anti-human CD68	biolegend	562111
Animal-free Blocking Solution	Cell Signaling Technology	15019
APC anti-human CD90	biolegend	559869
BD FACSDiva Software 8.0.2	BD Biosciences	657925
BD LSRFortessa	BD Biosciences	647177
BV605 anti-human CD11b	bidegend	563015
CellTiter 96 AQueous One Solution Cell Proliferation Assay(MTS)	Promega	G358A
Collagenase II	Solarbio	C8150
DAPI-containing Fluorescent Mounting Medium	Zhongshan Jingqiao Biotechnology	ZLI-9556
DAPI-containing Fluorescent Mounting Medium	Zhongshan Jingqiao Biotechnology	ZLI-9556
Dulbecco's Modified Eagle Medium (DMEM), High Glucose	HyClone	SH30022.01
Fetal bovine serum-Superfine (FBS)	Shanghai Zhongqiao Xindan Biotechnology Co., Ltd.	ZQ0500
FITC anti-human CD14	biolegend	325604
PBS, 1× (pH 7.4) Phosphate Buffered Saline	Servicebio	G4202
Penicillin-Streptomycin Mixture Solution (Double Antibiotics) 100×	Shanghai Zhongqiao Xindan Biotechnology Co., Ltd.	CSP006
QuickBlock Universal Protein Blocking and Antibody Dilution Buffer	Beyotime	P0270-500ml
Recombinant Human TNF-α	Peprotech	300-01A
Transcription Factor Staining Buffe	eBioscience	00-5523-00
Triton X-100	Solarbio	T8200
Trypsin Solution (EDTA-free)	Beijing Labgic Technology Co., Ltd.	BL526A