Primary porcine MAs were isolated using the methods described by Nobusue and Kano [5]. In brief, fat tissue was minced and digested in 0.1% (w/v) collagenase solution (Collagenase type II; Sigma–Aldrich) at 37 °C for 1 h with gentle agitation [12]. The digested cell suspension was then filtered through 150 and 250 μm nylon meshes (Kyoshin Rikoh), allowing the
ha tag to pass through but retaining unwanted stromal cells and tissue. The floating MAs in the top layer were collected and washed thrice by centrifugation. Isolated MAs were placed in 12.5 cm2 culture flasks (BD Falcon) filled with Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co.) with 20% (v/v) fetal bovine serum (Moregate BioTech). The flask was filled with the medium, turned upside down, and incubated in a humidified 5% CO2 atmosphere. Approximately 1 week later, the
cells had firmly attached themselves to the ceiling and developed fibroblast-like shape without any visible fat droplets. The medium was changed every 4 days until the cells had grown to semiconfluence at 14 days.
