Bs163

The omental tissue obtained from passage 3 mice was minced into 0.1 mm³ pieces. The samples were digested with 1 mg/ml of collagenase type Ⅰ (BS163, Biosharp, China) at 37 °C on a thermostat shaker at 150 rpm for 1 hour. After a 75 μm filtration, the suspension was centrifuged, and the cells were then plated in flasks.

The study was designed according to the Declaration of Helsinki and was approved by Ethic Committee of the Second Affiliated Hospital of Harbin Medical University (Ref: KY 2021-256). Written informed consent was obtained for 5 patients aging from 60 to 65 years old with OA, without rheumatoid arthritis, acute trauma, tumor, or infection of knee joint. The diagnosis of OA was based on clinical and radiological evidence of degenerative changes during surgery. The discarded specimens after total knee arthroplasty (TKA) were transferred into the biosafety cabinet within half an hour and washed twice with phosphate-buffered saline (PBS) solution to remove the blood, fat, and synovial fluid. The exposed cartilage tissue was cut into the final size about 0.5 mm × 0.5 mm, and put into 0.2% type II collagenase (Biosharp, China, BS163). After 4 hours, α-MEM medium (Cytiva, USA, SH30265.01) was added, with 10% fetal bovine serum (ExCell Bio, China, FSD500), 1% penicillin, and 1% streptomycin (Beyotime, China, C0222). Primary chondrocytes could be seen climbing out after about 7 days under optical microscope (ZEISS Axio Vert.A1, Germany). The medium was changed every 2 days and digested when the chondrocytes climbed to about 60% of the bottom. Chondrocytes used in this study were from passage 2 to 3 (P2-3).

Immediately following collection, tumor tissues and distal normal lung tissues were immersed in Hank’s balanced salt solution (HBSS, Gibco), and rapidly transported to laboratory at a low temperature. These samples were quickly divided into two pieces; one-half was treated with enzymatic digestion and cell sorting for scRNA-seq, and the other was fixed in 10% neutral formalin solution and paraffin-embedded for TRS, IHC and mIHC staining.
For preparing single cells, tissues were minced with scissors on ice and digested well with 1 mg/mL collagenase I (BS163, Biosharp), 0.5 mg/mL collagenase IV (BS165, Biosharp) and HBSS. After 30 min of incubation at 37°C on a shaker, samples were filtered using the 40μm strainers (Corning), centrifuged at 500 x g for 5 min at 4°C and then removed the supernatant. The remaining cell pellet was treated with red blood lysis buffer (Biosharp) for 5 min to remove the red blood cells, followed by centrifugation once more for 5 min (500g, 4°C). After the removal of the supernatant, the samples were resuspended in the sorting buffer (0.04% BSA and PBS). The dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec, Germany) according to the manufacture’s protocol. The single-cell suspensions with a rate of cell viability greater than 80% were handled according to manufacturer’s instructions subsequently for scRNA-seq.