Huc msc

Passage 2 generation of hucMSC was received from Cyagen Biotechnology Inc. (Item: HUXUB-01001 lot number: 130606L01, Cyagen Biotechnology Inc., Guangzhou, Guangdong, China). Cells were maintained in MSC NutriStem® XF Medium (Biological Industries, Beit HaEmek, Israel) containing 15% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) at 37℃ under 5% CO₂ in the air, replacing the culture medium every 2–3 days. hucMSC at passage 3 was used for subsequent experiments. The immunophenotype of the culture‐expanded hucMSC was characterized using flow cytometry based on previously described methods (Liu et al. 2012 (link)). All hucMSC showed positive expression for CD29, CD44, CD73, CD90, and CD105 and negative expression for CD11b, CD14, CD34, CD45, and HLA-DR. The adipogenic and osteogenic differentiation of hucMSC was assessed by Oil red O staining and Alizarin Red staining, respectively (Yuan et al. 2020 (link)). Human NP cells (HNPC) were isolated and cultured as previously reported (Dudek et al. 2017 (link)). After washing thrice with sterile phosphate-buffered saline (PBS), annulus fibrosus was meticulously eliminated from the human intervertebral disc tissues. Cells were cultured with media replacement every 72 h and passaged at 80%–90% confluence. HNPC at generation 2 or 3 was used herein.