Isolation and Culture of Primary Human Synovial Macrophages

Isolation and Culture of Primary Human Synovial Macrophages

I. Materials and Reagents

1. Human Synovial Tissue Samples 

Human synovial tissue samples are collected from surgical remnants following procedures such as joint replacement or arthroscopic examination. Samples should be promptly transported to the laboratory, preferably under refrigerated conditions at 4°C, and processed under sterile conditions. Note: Red choroidal hyperplastic synovial tissue contains more macrophages, and white tough synovium contains more fibroblasts.

1. Enzymatic Digestion and Cell Isolation Reagents

• Digestion enzyme mixture: Collagenase IV prepared at a concentration of 4 mg/mL in complete DMEM/F12 medium (the enzyme concentration may be adjusted according to tissue density).
• PBS (phosphate-buffered saline) supplemented with 0.5% BSA and 2 mM EDTA, utilized as the washing buffer for cell suspensions.

1. Magnetic Bead Separation System (Miltenyi Biotec beads) 

Use magnetic beads pre-coated with anti-CD14 antibodies specific for macrophage surface markers. Carefully review the manufacturer's instructions prior to use, ensuring the correct preparation of the magnetic stand, recommended cell concentration, and incubation time are strictly adhered to.

II. Experimental Procedure

1. Collection and Preliminary Processing of Synovial Tissue
2. During surgery, collect fresh human synovial tissue and immediately place it in pre-cooled PBS containing antibiotics. Minimize tissue exposure time to maintain cell viability.
3. Under a biosafety cabinet, use sterile scissors and forceps to remove visible blood vessels, connective tissue, and adipose tissue. Cut synovial tissue into small pieces of approximately 1–2 mm to enhance surface area for enzymatic digestion.

1. Enzymatic Digestion of Tissue
2. Transfer the finely chopped synovial tissue into a pre-prepared digestion enzyme mixture (4 mg/mL Collagenase IV in complete DMEM/F12 medium (DMEM + 10% FBS + 1% antibiotics)). Typically, add 10–15 mL of digestion solution per 1 mL of tissue.
3. Incubate the mixture in a 37°C shaking incubator with gentle agitation (60–80 rpm) for 5–8 hours. Monitor tissue dissociation every 2 hours.

1. Preparation and Pretreatment of Cell Suspension
2. Filtration: Pass the digested mixture through a 70 μm cell strainer to remove incompletely digested tissue fragments.
3. Centrifugation: Centrifuge the filtered cell suspension at 400 g for 10 minutes at 4°C to pellet the cells.
4. Washing: Discard the supernatant and resuspend the cells in pre-chilled PBS (containing 0.5% BSA), gently pipetting to achieve uniform suspension.
5. Repeat centrifugation and washing 1–2 times to remove residual digestion solution and cell debris.
6. Cell Counting: Count cells using trypan blue staining to ensure an adequate cell yield for subsequent magnetic bead labeling.

1. Magnetic Bead Labeling and Cell Sorting (According to Miltenyi Biotec Instructions: Appendix file)
2. Cell Pretreatment: Resuspend washed cells in an appropriate volume of MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) at a concentration of approximately 1×10^7 cells/mL.
3. Magnetic Bead Incubation: Add the recommended volume of pre-coated anti-macrophage (CD14) magnetic beads to the cell suspension, typically following the guideline of bead volume per 1×10^7 cells as stated in the manufacturer’s protocol. Incubate at 4°C or room temperature for 15–30 minutes with gentle mixing (specific incubation time according to literature and product instructions).
4. Washing: Dilute the cell suspension with MACS buffer post-incubation and centrifuge (300–400 g, 5 minutes) to remove unbound magnetic beads. Resuspend cells and repeat washing once if necessary to improve sorting purity.
5. Magnetic Separation: Place the resuspended cells in a magnetic separator stand, allowing labeled cells to adhere under magnetic attraction according to the system guidelines. Carefully discard the supernatant (unlabeled cells), then rinse and collect captured macrophages from the magnetic column. A second magnetic sorting can be performed to further enhance cell purity.

1. Validation and Culture of Macrophages

1)  Validate the purity of sorted cells using flow cytometry.

2)  Culture macrophages in complete DMEM medium supplemented with 25 ng/mL CSF to promote macrophage growth.