2.2. Isolation of ADSCs

Two weeks prior to laparotomy for biliary anastomosis, ADSCs were isolated from the lower abdominal subcutaneous adipose tissue of pigs as reported elsewhere [11,21]. Briefly, 20 g of minced subcutaneous adipose tissue were enzymatically digested with 0.1% collagenase (Collagenase NB 4G Proved Grade, SERVA Electrophoresis, Heidelberg, Germany) under shaking at 37 °C for 1 h. The stromal-vascular fraction was collected after centrifugation at 400× g for 5 min at room temperature. After filtration through a 40-mm strainer (BD Biosciences, Franklin Lakes, NJ, USA) and centrifugation at 1500× g for 5 min, the cells were cultured in complete medium (low-glucose Dulbecco's modified Eagle's medium, Wako Pure chemical industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO₂ at 37 °C. ADSCs were passaged every 5 days by seeding onto 100-mm culture dishes at a density of 1.1 × 10⁶ cells/dish until the third passage.