Isolation and Expansion of Adipose-Derived Cells

Adipose samples (n = 4) were harvested according to the Coleman technique [13 (link)] during scheduled abdominal liposuctions in patients undergoing breast reconstruction. The study was approved by the local Ethical Committee on Human Study (PROT.N. 3676/CE), and participants provided written informed consent to take part in the study. An average of 600 mg of adipose samples was washed in phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria), minced, and digested for 60 min in Dulbecco’s modified Eagle’s medium (DMEM; Euroclone Spa, Milan, Italy) containing 1.76 UI/mL collagenase solution (Roche Diagnostics GmbH, Mannheim, Germany) and 1% penicillin–streptomycin (P/S; 104 UI/mL and 10 mg/mL; PAA) at 37 °C with gentle agitation [14 (link)]. After enzyme inactivation by the addition of blocking media, the resulting cell suspension was centrifuged, filtered through a 100 μm cell strainer (BD Falcon, Durham, NC, USA), counted using 0.4% trypan blue (Biochrom AG, Berlin, Germany), and seeded in culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) at a density of 100,000 cells/cm² in Quantum 333 culture medium (PAA) as reported [11 (link)]. Once an 80–90% confluence was reached, cells were detached with 0.05% trypsin/0.02% EDTA (Euroclone), counted, and seeded at a density of 6000 cells/cm².