NP cell isolation and amplification

Rat NP cells were isolated from 23 Sprague–Dawley rats (male, 7–8 weeks old) according to a previous method [30]. Briefly, after NP tissue samples were separated, NP cells were isolated by digestion with 0.1% collagenase type II (Sigma–Aldrich, U.S.A.) for 4–6 h. All experimental rats were used according to the guidelines of the Ethics Committee at Yantai Yeda Hospital [YD (LU) 2014-0020]. The isolated NP cells were cultured in standard DMEM/F12 culture medium containing 10% FBS (Gibco, U.S.A.) in 95% humidity, 21% O₂, and 5% CO₂ at 37°C. When they were grown to 80% confluence, NP cells were subcultured. The passage 2 NP cells were seeded in the six-well culture plate and used for every test in the present study.