Type 2 collagenase solution

Nucleus pulposus samples from humans were obtained courtesy of the Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China). These samples were then thoroughly separated and segmented. Subsequently, a 1% type II collagenase solution (Beyotime) was used to digest the tissue suspension for 1 h. The cells were cultivated in Dulbecco’s modified Eagle’s medium (Corning) supplemented with 10% fetal serum albumin (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin and were incubated at 37°C in a 5% CO₂ environment. Primary cells were cultivated in 1% oxygen. During extended cell culture under normoxic conditions, the oxygen concentration was maintained at 20%. Clinicopathological data were acquired from the Second Affiliated Hospital, Zhejiang University School of Medicine. The study included patients who experienced disc injuries due to trauma. Ultimately, the study included 5 young and 5 elderly patients (Table S1). Informed consent was obtained from all participants. The human studies adhered to the Declaration of Helsinki principles and received approval from the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China).

Rat chondrocytes were isolated from the cartilage tissues of SPF Wistar rats weighing 180 ± 20 g, as described previously [34 (link)]. Briefly, the rats were killed by cervical dislocation, and cartilage tissues from the knee were collected under sterile conditions. After washing thrice with PBS, the cartilage tissues were cut into small pieces (1 mm³), and then 0.2% type II collagenase solution (Beyotime Biotechnology, Shanghai, China): 0.25% trypsin solution (Beyotime Biotechnology; v/v, 4:1; including DNase) was added. After digestion at 37 °C for 6 h, DMEM/F12 medium (Thermo Fisher Scientific) was added to terminate the digestion. After filtering, the collected chondrocyte suspension was centrifuged at 1000 rpm for 5 min, and the chondrocytes were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Afterwards, the chondrocytes were inoculated into a 25 cm²-cell culture plate and passaged when the cell confluence reached 80–90%.