Isolation and culturing of NP cells

Human NP tissues were carefully isolated, washed three times with PBS, cut into 1 mm³ pieces with ophthalmic scissors, and placed in a 15-mL centrifuge tube. At the temperature of 37 °C, NP tissues were digested for 40 min in PBS solution with 0.25% trypsin (Gibco-BRL, Grand Island, NY). The digestive solution was removed, and the left tissues were washed with PBS again. Next, NP tissues were digested for 4 h in PBS solution with 0.025% type II collagen (Invitrogen, USA), followed by filtering, centrifugation, and discarding the upper supernatant. Then, the NP cells were resuspended in DMEM/F12 supplemented with 15% fetal bovine serum (FBS, Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin, and 1% l-glutamine, and incubated at 37 °C in an atmosphere containing 5% CO₂. At 80–90% confluence, cells were digested by 0.25% trypsin solution and subculture in DMEM/F12 supplemented with 15% FBS, 100 μg/mL streptomycin, 100 U/mL penicillin at 37 °C in a humidified 5% CO₂ atmosphere. The culture medium was replaced twice every week. The second passage cells were used for subsequent experiments.