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^6,7. Among these, synovitis represents the earliest and most central pathological hallmark of RA. Fibroblast-like synoviocytes (FLS), residing as the predominant resident cells of the synovium, are pivotal in orchestrating both synovial inflammation and bone destruction^8,9. During the synovial inflammatory response in RA, Rheumatoid arthritis Fibroblast-like synoviocytes (RA-FLS) lose their normal "regulatory" phenotype and instead exhibit a "pro-inflammatory" profile¹⁰. This shift leads to the massive production of inflammatory cytokines, which may activate related energy metabolic pathways, leading to increased cell proliferation and further amplifying the synovial inflammatory cascade^11,12,13. Simultaneously, RA-FLS interact with other immune cells (such as macrophages, T cells, and B cells) to sustain a persistent chronic inflammatory state^14,15. Furthermore, RA-FLS exhibit cancer-like characteristics, including enhanced migratory capacity, invasiveness, uncontrolled proliferation, and resistance to apoptosis, and are actively involved in the processes of cartilage and bone erosion¹⁶.

In recent years, FLS have been widely utilized as in vitro models for studying joint inflammatory diseases such as RA, a trend driven in part by the limitations of current first-line pharmacotherapies^17,18,19. They are employed in 2D cultures, 3D models, and organ-on-a-chip systems to investigate disease mechanisms, validate therapeutic targets, and conduct drug screening^20,21,22,23. Furthermore, RAFLS serve as a foundation for advanced applications, including patient-derived synovial organoids for predicting DMARDs response; genetic editing via CRISPR/Cas9 to study gene function in RA progression; and as a cell source for engineering functional synovial tissue grafts^24,25,26.

Obtaining short-term cultured RA-FLS from the synovial tissue of rheumatoid arthritis patients serves as the initial step for many experiments. Compared to FLS cell lines, short-term cultured, patient-derived RA-FLS retain inter-patient heterogeneity and disease-specific characteristics, enabling more authentic representation of disease-related transcriptional factors and pathway regulatory differences. Consequently, cell yield, growth rate, and functional performance may be influenced by patient-specific factors such as age, disease duration, medication history, and disease activity, leading to a certain degree of inter-batch variability. Researchers should therefore evaluate the clinical background of the tissue source prior to use and conduct experiments within early passages (P3-P7) to maintain phenotypic stability of the cells.

This study details a streamlined protocol for extracting and identifying RA-FLS from knee synovial tissue. The method employs sequential enzymatic digestion (trypsin followed by type II collagenase) combined with gentle mechanical dissociation. Evidence suggests that such optimized, multi-step enzymatic strategies generally outperform single-enzyme or non-sequential mixed-enzyme treatments, which may compromise cell viability and phenotype^27,28,29,30. By comparison, the presented protocol enables more efficient and gentle isolation of high-viability, high-purity RA-FLS, minimizing cellular damage and phenotypic loss to ensure suitability for downstream functional assays.

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 the Table of Materials.

1. Acquisition and preparation of synovial tissue

NOTE: Place the synovial tissue obtained during surgery in a sterile container filled with 0.9% saline solution or PBS to prevent contamination during transfer from the operating room to the biosafety cabinet. Use ethanol immersion followed by washing with a penicillin-streptomycin solution as critical steps to prevent bacterial or fungal contamination, ensuring the success of subsequent experiments.

1. Immerse fresh synovial tissue into a 50 mL centrifuge tube containing PBS and transfer it to a biosafety cabinet.
2. Transfer the synovial tissue to a Petri dish containing 75% ethanol and immerse for 30 s for disinfection.
3. Remove fat and bone tissue from synovial tissue using sterile surgical scissors and forceps, and then transfer the remaining tissue to a new culture dish.
4. Wash the obtained synovial tissue 3 times with PBS containing 10% penicillin/streptomycin.

2. Digestion of synovial tissue

NOTE: Use trypsin to disrupt cell junctions and the extracellular matrix within synovial tissue for subsequent isolation.Apply type II collagenase to selectively degrade the extracellular matrix structure, facilitating cell dissociation from the matrix.Sterilize all surgical scissors, forceps, and cell strainers required for synovial tissue dissection by autoclaving prior to use. After digestion, the tissue fragments should appear loosened, with frayed edges, and the medium may become slightly turbid. Undigested tissue will remain compact and opaque.

1. Mince the tissue repeatedly with scissors into 1mm × 1mm fragments.
2. Add 0.25% trypsin to the tissue and digest at 37 °C in a constant temperature shaker (at 100-150 rpm) or in a cell incubator for 30 min.
3. Prepare a 10% fetal bovine serum (FBS) solution using Phosphate-Buffered Saline (PBS).
4. Transfer the tissue into 15 mL or 50 mL centrifuge tubes according to the amount of tissue, and neutralize and digest it with PBS containing 10% fetal bovine serum (FBS).
5. Centrifuge (4 °C, 500 × g, 5 min), then discard the supernatant and retain the pellet.
6. Resuspend the pellet in PBS.
7. Centrifuge (4 °C, 500 × g, 5 min), then discard the supernatant and retain the pellet.
8. Add 100 mg of type II collagenase to 50 mL of DMEM medium to prepare a 2 mg/mL type II collagenase solution.
9. Filter the prepared type II collagenase solution through a 0.22 µm membrane for sterilization.
10. Add 2 mg/mL type II collagenase and digest in a 37 °C constant temperature shaker (at 100-150 rpm) or cell incubator for 4 h.

3. Isolation and culture of fibroblast-like synoviocytes

NOTE: Perform all cell culture procedures on a biosafety cabinet under sterile conditions and wear sterile gloves to prevent contamination.

1. Prepare complete DMEM medium by supplementing DMEM basal medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
2. Place a 100-mesh cell strainer on a sterile Petri dish and pour the digested synovial tissue and fluid onto the strainer.
3. Transfer the cell strainer with residual tissue to another sterile Petri dish.
4. Grind the remaining tissue on the strainer using the end of a 20 mL sterile syringe plunger rod until only white, fibrous remnants remain, and no further cell suspension can be expressed.
5. Rinse the strainer with an appropriate volume of complete DMEM medium.
6. Collect all filtrates, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
7. Resuspend the pellet in PBS, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
8. Resuspend the cells in complete DMEM medium.
9. Perform cell counting and transfer the cells to a culture flask.
10. Incubate the flask in a cell culture incubator (37 °C, 5% CO₂).
11. Replace the medium after 48 h.
12. Digest the cells with trypsin and passage at a 1:3 ratio when they reach full confluency.

4. Identify RA-FLS using vimentin immunofluorescence staining

NOTE: Pre-warm the PBS solution used for cell washing in a water bath set at 37 °C. All reagent volumes specified in this section are standardized for a 48-well plate format. Volumes should be scaled proportionally based on the growth area when using plates of a different format.

1. Digest cells with trypsin, resuspend the harvested cells, and dilute to 1 × 10⁴ cells/L using complete DMEM medium.
2. Seed RA-FLS into a 48-well plate at 200 µL per well.
3. Place in a cell culture incubator (37 °C, 5% CO₂) and incubate for 24 h.
4. Discard the medium and wash the cells three times with pre-warmed PBS.
5. Add 4% paraformaldehyde to fix cells for 10 min, discard the fixative, and wash three times with PBS.
6. Prepare 150 µL of 0.1% Triton X-100 solution in PBS per well. Ensure complete mixing by vortexing or gentle inversion before use.
7. Add 0.1% Triton X-100 solution to the wells and incubate for 10 min to permeabilize the cells.
8. Discard the 0.1% Triton X-100 solution, and wash three times with PBS.
9. Add 150 µL of animal-free blocking solution per well, incubate at room temperature for 1 h.
10. Discard the blocking solution. Add 150 µL of vimentin primary antibody working solution (diluted 1:200 in antibody diluent) to each well.
11. Incubate with primary antibody at 4 °C overnight.
12. Discard the primary antibody and wash the cells five times with PBS.
13. Add 150 µL of Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (diluted 1:50 in universal antibody diluent) per well, and incubate at 37 °C for 1 h protected from light.
14. Discard the secondary antibody and wash the cells three times with PBS.
15. Add one drop of DAPI-containing antifade mounting medium per well, and dry for 10 min in the dark.
16. Observe and capture images using an inverted fluorescence microscope equipped with a 20× objective and appropriate filter sets for DAPI and FITC/GFP.
    NOTE:Wash cells with PBS by completely filling the wells with PBS, incubating for 5 min, and then removing the PBS. This procedure constitutes one wash cycle.

5. Quantify RA-FLS and assess purity using flow cytometry

NOTE: Use Vimentin and CD90 as positive markers to identify RA-FLS, while applying CD68, CD14, and CD11b as negative controls to exclude immune cell contamination. Vimentin serves as an intracellular-specific intermediate filament protein predominantly localized in the cytoplasm of RA-FLS. CD90 functions as a critical surface marker highly expressed on RA-FLS, enabling their distinction from immune cells. While CD68, CD14, and CD11b are specifically expressed in macrophages, monocytes, and myeloid cells, they are employed as negative markers to exclude immune cell contamination. Assign fluorochromes to cell surface markers based on their binding specificity, and ensure no overlap in fluorescence channels. For example, in this experiment, CD90 and CD68 share the same fluorochrome; therefore, do not add them to the same centrifuge tube. Prior to incubating with vimentin antibody, add fixation and permeabilization agents to disrupt the cell membrane. If the flow cytometer has sufficient fluorescent channels, markers such as CD90 and CD68 can be assigned to different channels for multi-color simultaneous analysis to further improve detection efficiency and information content.

1. Digest third-generation RA-FLS using EDTA-free trypsin.
2. Terminate digestion with complete DMEM medium, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
3. Resuspend the pellet in PBS, perform cell counting, and adjust the cell concentration to 1 × 10⁶ cells/mL using PBS.
4. Distribute the cells into three centrifuge tubes, adding 200 µL of cell suspension to each tube.
5. Add CD11b antibody (diluted 1:100 in universal antibody diluent) at a final volume of 100 µL to Tube 1. Vortex to mix.
   1. After 15 min incubation, add 1 mL fixation/permeabilization agent to Tube 1, vortex to mix, and incubate for 30 min protected from light.
   2. Add 1 mL fixation/permeabilization buffer to Tube 1 to terminate permeabilization, vortex to mix, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
   3. Prepare 100 µL of vimentin and CD68 antibody cocktail (each diluted 1:100 in universal antibody diluent), add to Tube 1, vortex to mix, and incubate protected from light for 30 min.
   4. Add 1 mL fixation/permeabilization buffer to Tube 1, vortex to mix, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
   5. Add 100 µL of Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (diluted 1:50 in universal antibody diluent) to Tube 1. Vortex to mix and incubate protected from light for 30 min.
   6. Add 1 mL of Buffer to Tube 1, vortex to mix, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
   7. Add 300 µL of Buffer to Tube 1, and vortex to mix for the loading sample.
6. Add CD90 and CD14 antibodies (each diluted 1:100 in universal antibody diluent) to Tube 2 to a final volume of 100 µL. Leave Tube 3 without antibodies as a negative control.
   1. After 15 min incubation, add 1 mL PBS to Tubes 2 and 3.
   2. Centrifuge tubes 2 and 3 (4 °C, 500 × g, 5 min), discard the supernatant, add 300 µL of Buffer, and vortex to mix for the loading sample.
7. Perform flow cytometry acquisition of the prepared samples.
8. Analyze the flow cytometry data and perform statistics using compatible software.

6. Measuring TNF-α-induced RA-FLS proliferation using the MTS assay

1. Digest third-generation RA-FLS using EDTA-free trypsin.
2. Terminate digestion with complete DMEM medium, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
3. Resuspend the pellet in complete DMEM medium, perform cell counting, and dilute to 1 × 10⁵ cells/L.
4. Seed the resuspended cells into a 96-well plate at 100 µL per well.
5. Incubate the plate in a cell culture incubator until cell adhesion occurs, then divide into blank control and intervention groups.
6. Add 10 ng/mL TNF-α to the intervention group and an equal volume of PBS to the blank control group.
7. After 24 h of intervention, add 10 µL MTS reagent to each well and continue incubation in the cell culture incubator.
8. After 1 h of incubation, measure the absorbance (OD) at 490 nm using a microplate reader.
9. Calculate cell proliferation viability based on the OD value of each well.

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