Collagenase 1 solution

hASCs were isolated from lipoaspirate by the enzymatic digestion method. In brief, liquid adipose tissue was washed with phosphate buffer saline (PBS) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.2 mg/ml amphotericin-B (Sigma–Aldrich). Washing of the sample was followed by enzymatic digestion with the collagenase-I solution (Gibco, USA) in low glucose Dulbecco's modified Eagle's medium (LG-DMEM; Sigma Aldrich, USA). After centrifugation at 1200 rpm for 5–7 min, the resulting stromal vascular fraction (SVF) was passed through a 100 μm cell strainer.

The hADSCs were isolated from adipose tissue, cultured for differentiation, and stained with the Oil Red O according to our previous study.31 (link) Briefly, 10 grams of fresh SATs were washed in physiological saline (PBS) three times, cut into a size of 1 mm × 1 mm, and digested in collagenase I solution (Gibco, Life Technology, China) for 1 hour at 37°C. After that, the mixture was filtered through a 70-µm cell strainer (BD Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged at 150× g for 10 minutes. Next, the supernatant was gently poured off and the cells were combined with 3 mL of erythrocyte lysate (Beyotime Institute of Biotechnology, Shanghai, China) and then incubated for 3 minutes and centrifuged at 150× g for 10 minutes to collect the cells. The cells were washed with PBS three times through centrifugation at 150 g for 10 minutes each and finally cultured in DMEM/F12 (Life Technologies, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (FBS; Life Technologies). The mature adipocytes that were induced from MSL hADSCs were stimulated by 1 μM, 5 μM, or 10 μM of isoprenaline (Hefeng Pharmaceutical Co., Ltd., Shanghai, China).

Approximately 10g of SATs were first washed with phosphate buffer saline (PBS) for three times. Then the sample underwent fragmentation into fragments less than 1 mm³, and digested at 37°C with 0.1% (w/v) collagenase I solution (Gibco, Life Technology, China) for 1.5 hours. The digestion was terminated with DMEM/F12 (Gibco, Life Technology, China) and filtered by 100μm cell strainer (NEST Biotechnology Co., Ltd., China), centrifuged at 800 rpm for 5 min, gently poured out the supernatant. Subsequently, 3mL red blood cell lysis buffer (Merck, China) was used to remove red blood cells, and after centrifuged at 800 rpm for 5 min again, the isolated cells were suspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technology, Australia), 100U/mL penicillin and 100 μg/mL streptomycin were added to the culture medium. The cells were then incubated at 37°C in an environment containing 5% CO₂. In order to induce the hADSCs differentiated to adipocytes, when hADSCs grew to almost 100% confluency, the hADSCs were cultured in complete DMEM/F12 medium which contained 1μM dexamethasone, 10μM insulin, 0.5mM isobutylmethylxanthine (IBMX), and 200μM indomethacin (four reagents were purchased from Merck, China); the culture medium was replaced every 48 hours.