10 n naoh

Nonwoven-PGA polymer mesh (0.3 mm × 150 mg/cc, 20 cm × 30 cm sheet, BIOFELT) scaffold was cut into 5 mm × 5 mm squares as described previously [43 (link)]. PGA squares were treated with 1.0 N NaOH (SigmaAldrich) for 1 minute, washed in distilled water, sterilized with 70% ethanol for 30 minutes, and air-dried under sterile conditions overnight. On the next day, PGA squares were treated with human collagen type IV (20ul/mL, SigmaAldrich; xenogeneic-free condition) or 0.1% porcine skin-derived gelatin (SigmaAldrich; xenogeneic condition) at 37°C for 1 hour, air-dried for 2–3 hours under sterile conditions, and moved into a 24-well low attachment dish (Corning).
Prior to generation of engineered vascular tissues, XF-hiPSC-VSMCs-P or XG-hiPSC-VSMCs-P cultured in the xenogeneic-free or xenogeneic collagen promoting medium for 5 days were harvested using TrypLE or 0.05% Trypsin-EDTA, respectively. Cells were then resuspended in collagen promoting medium at the density of 10 million cells/mL. 40 μl of the cell suspension was dropped onto the PGA mesh and then incubated at 37°C and 5% CO₂ for one hour to allow the cells to adhere. The wells were then filled with 1 mL of xenogeneic-free or xenogeneic collagen promoting medium. The medium was changed every other day for 21 days, and then the tissues were harvested for further analysis or implantation.