Collagenase 2

Primary NP cells were isolated and cultured as previously reported (5 (link)). The IDD and control NP tissue samples were washed three times with D-Hanks solution under aseptic conditions. These specimens were cut into 1-mm³ pieces, and digested with 0.25% trypsin (Beyotime Institute of Biotechnology) and 0.2% collagenase II (Beyotime Institute of Biotechnology) for 3 h at 37˚C. NP cells were filtered through a 200-mesh sieve, washed three times with PBS and the supernatant was discarded following centrifugation at 2,000 x g for 5 min (37˚C). Cells were washed with DMEM-F12 (Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% FBS to terminate digestion. After centrifugation at 2,000 x g for 5 min (37˚C), NP cells were counted and seeded into 25 cm² culture dishes. DMEM-F12 medium was supplemented with 15% FBS (Gibco; Thermo Fisher Scientific, Inc.), 10 µg/ml insulin and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) was used to culture NP cells under conventional incubation conditions (37˚C, 5% CO₂ and 95% humidity), and the medium was refreshed twice a week. When NP confluency reached 80%, cells were passaged at a ratio of 1:2. Cells in passage P2 were used for subsequent experiments.

A total of 20 male Sprague-Dawley rats (weight, 200-220 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All Sprague Dawley rats were reared under specific pathogen-free conditions. Rats were housed under laminar flow in an isolated room with controlled temperature and at a 12-h light/dark cycle. Food and water were available ad libitum. The rats were sacrificed by injecting 100-200 mg/kg pentobarbital sodium at the end of the experiments. Death of rats was confirmed by observation of respiration and heartbeat. Primary chondrocytes were isolated from the bilateral hip joints of 4-week-old male rats. The cartilage of the rat hip joint was cut into 1 mm³ pieces in a sterile manner and then treated with 0.25% (V/V) trypsin/EDTA (cat. no. C0201; Beyotime Institute of Biotechnology) for 1 h and digested with 0.2% (V/V) collagenase II (cat. no. C2-28; Sigma-Aldrich; Merck KGaA) in DMEM-F12 at 37˚C in an atmosphere with 5% CO₂ for 6 h. Next, the suspensions were centrifuged at 1,609 x g at room temperature for 5 min and cultured at 37˚C under 5% CO₂ in DMEM-F12 with 10% (V/V) FBS, 1% (V/V) penicillin and streptomycin. Primary chondrocytes from the first passage were used for in vitro experiments.