Type 2 collagenase

Immediately after surgical resection of the tumor, several tissue blocks (approximately 0.5 cm) were removed from the center of the tumor mass to avoid blood vessels. The tissue blocks were placed in RPMI1640 medium (iCell, Shanghai, China), sealed and stored at 4 °C, and transported to the laboratory within 1 h. Tumor tissues were washed three times with D-Hanks solution (Solarbio, Beijing, China) and dissected into 1 mm³ samples in a culture dish. The tissue was mixed with 5 mL type II collagenase (final concentration of 1 mg/mL; Solarbio), followed by digestion for 1.5 h in a 37 °C water bath (Figure 7C). Digestion was terminated by adding 5 mL culture medium and centrifugation at 1000 rpm for 5 min. The cell suspension was filtered twice through a 200-mesh filter. Finally, the RPMI1640 complete medium was added, and the cell suspension was transferred to T25 culture flasks. The complete medium was supplemented with 15% FBS (iCell), 1% penicillin-streptomycin solution (iCell), 1× L-glutamine (iCell), and 20 ng/mL basic fibroblast growth factor (bFGF, iCell). During the culture period, cells were incubated in a humidified incubator at 37 °C with 5% CO₂, and the medium was changed every 2 d. The primary cell line was named PriGIST, and the PriGIST cells were divided into two parts: one was introduced with genes for immortalization, and the other was used as a control.

Synovial tissues from patients with RA (Patient No. R41 to No. R50, n = 10) or OA (Patient No. 41 to No. O50, n = 10) were minced into small pieces and digested for 4 h at 37°C and 5% CO₂ in 3 ml of DMEM containing 4% type II collagenase (Solarbio, China) until the tissue pieces were dispersed into a cell suspension. The cell suspension was filtered through a 70 μm cell strainer and resuspended in DMEM containing 10% FBS. Synovial fibroblast cells were incubated at 37°C in a humidified incubator containing 5% CO₂. Cells that passed for 3–8 generations were used in subsequent experiments.

OA and normal cartilage tissues were obtained as discarded specimens from the hospital, and divided into superficial and middle layers within a 2-hour timeframe expeditiously. Subsequently, the collected cartilage was subjected to enzymatic digestion using 0.2% type II collagenase (Cat# C8150, Solarbio) in DMEM (Cat# 31600034, Solarbio) at 37°C for 3 hours. The resulting cells were then filtered through a 70-μm nylon cell strainer and harvested via centrifugation at 250 g for 5 minutes. The cells were resuspended in DMEM culture medium enriched with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). These cells were then seeded in 60 mm diameter culture dishes, with the culture medium being refreshed every 3 days to maintain optimal conditions for cell growth and viability.

NRCMs were obtained from newborn rats (1–2 days after birth). Briefly, heart tissues were collected from newborn rats and washed with D-Hank’s solution. Subsequently, the heart tissues were minced prior to digestion with trypsin (0.075%; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, United States) and type II collagenase (0.1%; Solarbio, Beijing, China). To suspend the cells, high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific) containing 100 U/mL of penicillin, 100 mg/mL streptomycin (Gibco, Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel) was added. Then, cells were seeded on 10-cm plastic dishes in a humid incubator for a 1.5-h period under 5% CO₂ and 37°C conditions. DMEM-suspended cells were inoculated in glass-bottom cell culture dishes or 48-well plates at 20,000 cells/well in 5% CO₂ in a humidified atmosphere at 37°C.

Primary chondrocytes were obtained from male C57BL/6 mice aged 4 weeks. The femoral head was removed and cut into pieces, digested with trypsin for 40 min and then digested with type II collagenase for 8 h. The isolated chondrocytes were confirmed by toluidine staining (Solarbio, Beijing, China) and western blot of type II collagen (1:200, Abcam, Cambridge, UK) (Additional file 1: Fig. S1). Primary chondrocytes were cultured at F-12/DMEM (Invitrogen, Thermo Fisher Scientific, USA) supplemented with 10% FBS and 1% penicillin–streptomycin–gentamicin solution. Then, 10 nM IL-1β and 10 nM TNF-α (PeproTech Inc., Rocky Hill, NJ, USA) were used to induce inflammation in cultured primary chondrocytes.