L glutamine

Human adipose tissue was obtained from the First Affiliated Hospital of Dalian Medical University, China. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Dalian Medical University (approval No. PJ-KS-KY-2020-54) on March 7, 2017. All participants who agreed to donate adipose tissue signed informed consent. hADSCs were isolated as previously described (Han et al., 2014a). Cells were maintained in basal medium (Cyagen, Guangzhou, China) supplemented with 10% fetal bovine serum (Cyagen), 1% L-glutamine (Cyagen) and 1% penicillin/streptomycin solution (Cyagen), and cultured at 37°C in a humidified incubator containing 5% CO₂. hADSCs adhered to the plastic and proliferated. The medium was changed every 2 days unless indicated otherwise. The morphology of purified cells was examined by microscopy. Cells at passage 5 were used for the experiments.

For osteogenic differentiation cells were firstly grown to 100% confluence, and then cultured with medium containing α-MEM with 10% FBS, ascorbate, β-glycerophos-phate and dexamethasone (Cyagen Biosciences, Jiangsu, China) for nine days. Medium was changed every 3 days. Alizarin red staining was performed and recorded using an inverted microscope (Olympus, Tokyo, Japan). In addition, mRNA expression of Runx2 and osteocalcin (OCN) was analyzed with qPCR. To assess adipogenic differentiation cells were cultured at 100% confluence for 2∼3 weeks in α-MEM containing 10% FBS, L-glutamine, insulin, isobutylmethylxanthine, rosiglitazone and dexame-thasone (Cyagen Biosciences, Jiangsu, China). Medium was changed every 2∼3 days and then Oil red staining was performed. For chondrogenic differentiation, 3−4×10⁵ suture mesenchyme cells were cultured as a pellet at the bottom of a 15 ml aseptic tube in 0.5 ml α-MEM medium supplemented TGF-β3, dexamethasone, ascorbate, sodium pyruvate and proline (Cyagen Biosciences, Jiangsu, China) and changed every 3 days. After 21 days culture, the cell microsphere was formalin-fixed and paraffin-em-bedded and then sectioned at 4 mm, stained with Alcian blue.