Isolation and Culture of Intervertebral Disc Progenitor Cells

Primacy NPCs were isolated from human intervertebral disc (IDD patients and controls), mouse intervertebral disc (miR-150-5p KO and C57BL/6 wild-type mice). NPCs were preserved as a monolayer in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FCS), 100 IU/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% carbon dioxide at 37 °C. All the experiments involved the use of human NPCs at a confluence rate of 85%. As previously described [10 (link)], the cells were inoculated into 96-well plates for three-dimensional culture of the NPCs. The NPCs were then cultured at 37 °C and 5% carbon dioxide in 200 µl of NPC growth medium. After 21 days of culture, the cells were collected for further evaluation. Using a Silencer® siRNA labeling kit or miRNA negative control (mirVana miRNA mimics/inhibitor negative control #1) (Life Technologies), Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA), and Cy3-labeled or unlabeled miR-150-5p (a mirVana miRNA mimic or inhibitor) were transfected into human NPCs, SW1353 cells, or C28/I2 cells at 50 nM. The FBXW11 expression plasmid (pcDNA 3.1/V5-His TOPO TA Expression Kit) (Invitrogen) was subsequently obtained.