Cell strainer

Mouse primary RTECs (pRTECs) culture was performed as previously described 24 . Briefly, the cortical part of the kidneys from 4-week-old Dusp2^fl/fl mice or AAV9-Dusp2 mice was collected, minced, and transferred to 10 mL HBSS (Hank's balanced salt solution). 0.1% collagenase II was added for 8 min, and then the digestion was terminated using the culture medium. Cells were filtered using a cell strainer (70 μm, Beyotime), and the suspension was centrifuged at 1000 rpm for 3min. These renal tubules were collected after an ultracentrifugation density gradient. After being washed with PBS, the precipitate was resuspended in DMEM/F12 (1:1) medium containing 10% FBS for 4-5 days.

Nucleus pulposus cells were isolated from different donors and these cells were used for the cell-based in vitro experiments in this study. The isolation process of NPCs was performed as described previously (Zhang et al., 2018 (link)). Nucleus pulposus tissue samples were washed three times with PBS, then cut and digested using 0.25% trypsin (Servicebio, Wuhan, China) and 0.01% EDTA at 37°C for approximately 45 min. Next, tissue pieces were digested by type II collagenase (Servicebio) for 4 h at 37°C. The cell suspension was sieved through a cell strainer (70 μm; Beyotime, Shanghai, China) and centrifuged at 1,000 × g for 5 min. NPCs were resuspended in DMEM/F12 with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Beyotime) and cultured in a humidified atmosphere of 5% CO₂ at 37°C, changing medium every 3 days. Cells at second passage were used for subsequent experiments. To establish the degeneration model of NPCs, cells were incubated for 24 h then treated with Recombinant Human Interleukin 1β (IL-1β) (10 ng/mL, Beyotime) for 24 or 48 h. Total RNA or protein were then extracted (Bai et al., 2019 (link); Zhang et al., 2019 (link)).