Isolation and Culture of Intervertebral Disc Nucleus Pulposus Cells

This method has been reported in our recent study [2 (link)]. Briefly, the collected intervertebral disc tissue was transported to laboratory using 0.9% sterile saline within two hours. Then, the tissue was washed 3 times using PBS (Servicebio, Wuhan, China) in super clean bench. Next, gel-like NP tissue was separated and digested using 0.25% Trypsin-EDTA (C0209-100 mL, Beyotime, Shanghai, China) for 30 minutes and 0.2% collagenase type II (Invitrogen, USA) for another 1 h at 37°C under a shaker (70 r/min). Finally, the isolated NP cells were resuspended in complete culture medium (Gibco; Thermo Fisher Scientific, Inc.) and cultured in a 37°C incubator. At the third, half of the culture medium will be replaced by complete medium. Five days after isolation, the spindle-shaped NP cells will move out and adhered at the bottom which we called passage 0. When the cell density reaches about 80%, the NP cells could be used further experiments.