Collagenase type 1 solution

Human ASC were isolated from human subcutaneous adipose tissue samples obtained from liposuction procedures as previously described 19. Samples were digested in 1 mg/ml of collagenase type I solution (Worthington Biochemical, Lakewood, NJ, USA) for one hour at 37°C and centrifuged at 300 g for 8 min. to separate the stromal cell fraction (pellet) from adipocytes. The pellets were filtered through 250 μm Nitex filters (Sefar America Inc., Kansas City, MO, USA) and treated with red cell lysis buffer (eBiosciences, San Diego, CA, USA). The final pellet was resuspended and cultured in EGM‐2MV (Lonza, Walkersville, MD, USA). Media were changed after 24 hrs and then every 2–3 days. ASC were passaged when 60–80% confluent and used at passages 3–5.

Primary HDFs were derived from foreskins of three 9 to 12 year-old boys after circumcision. Written informed consents were obtained from parents of all subjects. The samples were aseptically collected and washed several times with 75% alcohol and phosphate buffered saline (PBS) containing 1% antibiotic-antimycotic solution (PAA, Pasching, Austria). After removing the epidermis, the pure dermis was cut into small pieces and transferred into a falcon tube containing 0.03% collagenase type I solution (Worthington Biochemical Corporation, Lakewood, NJ, USA). The pure dermis was digested in an incubator shaker at 37°C for 6–12 h. Cells were then rinsed with PBS before being cultured in Dulbecco Modified Eagle Medium (DMEM, Flowlab™, North Ryde, Australia), 10% fetal bovine serum (PAA, Austria), 10,000 μg/mL penicillin/streptomycin (Gibco, Grand Island, NY, USA), 250 μg/mL amphotericin B (PAA, Austria), 100 mg/mL gentamycin (PAA, Austria) and incubated in 5% CO₂ atmosphere at 37°C. This research has been approved by the National University of Malaysia Ethical Committee (Approval Project Code: FF-287-2009).

Isolation and initiation of human ASC cultures were performed as described [18 (link)]. As source served abdominal subcutaneous fat tissue derived from plastic surgeries, generously provided by Dr. Ulrich Rieger (Klinik für Plastische und Ästhetische Chirurgie, Wiederherstellungs und Handchirurgie, Markus Krankenhaus, Frankfurt/Main, Germany). The fat tissue was placed in PBS with 2% penicillin/streptomycin (Biochrom, Berlin, Germany) incubated overnight (4 °C). On the next day, skin and blood vessels were mechanically removed by scissors and forceps. Small pieces, with approximately 5 mm lengths, were given to a collagenase type I solution (Worthington, Lakewood, USA) and incubated for 3 h at 37 °C. Cell debris was discarded by filtration through sterile gauze. Then, the cell suspension was centrifuged (400× g, 6 min, 4 °C), the cell pellet resuspended in medium, and passed through a cell strainer (70 µm, Greiner, Frickenhausen, Germany). Next, cells were separated by density filtration using a Biocoll solution with a specific density of 1.077 g/mL. After another centrifugation (400× g, 30 min, 4 °C), ASCs were isolated from the opaque interphase and seeded in DMEM supplemented with 1% UltroSerG (Pall, Dreieich, Germany) and 1% penicillin/streptomycin. The medium was renewed every 3 days.