The NPCs were isolated using the method as previously described [4 (link)]. First, the collected NP tissues were cut with a size of 1 mm3 and incubated with trypsin (0.25%, PB180228, Procell, Wuhan, China) for 30 min, followed by centrifugation at 1,000g (E2658, Beyotime, Shanghai, China) for 10 min and incubation with collagenase type II (40508ES60, Qcbio Science & Technologies Co., Ltd, Shanghai, China) at 37°C for 4 h. Later, the treated NP tissues were filtered with a 200-mesh filter (S4203, Aladdin, Shanghai, China), maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F medium (with the inclusion of 1% penicillin–streptomycin mixture solution, PM150312A, Procell, Wuhan, China) supplemented with 20% of fetal bovine serum (164210, Procell, Wuhan, China) and cultured in an incubator (BC-J80, BoXun, Shanghai, China) at 37°C with 5% CO2. The cell medium was replaced 2–3 times a week.
When the NPCs grew attached, the morphology of primary NPCs was observed (magnification 200×) under an inverted microscope (Ts2-FL, Nikon, Tokyo, Japan) as appropriate. NPCs with passage ≤3 (P ≤ 3) were used for the subsequent experiments in the present study [22 (link)–24 (link)].
When the NPCs grew attached, the morphology of primary NPCs was observed (magnification 200×) under an inverted microscope (Ts2-FL, Nikon, Tokyo, Japan) as appropriate. NPCs with passage ≤3 (P ≤ 3) were used for the subsequent experiments in the present study [22 (link)–24 (link)].