Isolation of Stromal Vascular Cells from Human Adipose Tissue
1. Adipose tissue was obtained by liposuction from abdominal, hip, and thigh regions of healthy female donors aged 18–39.
2. The stromal vascular fraction (SVF) was separated from adipose tissue. Lipoaspirate (300–400 ml) was washed with Hanks' balanced salt solution containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin, Life Technologies-BRL) and 2.5 μg/ml amphotericin B.
3. Washed adipose tissue was digested for 2 h on a shaker at 37°C in HBSS containing 0.2% collagenase.
4. Floating adipocytes were aspirated from pelleted SVF cells after centrifugation at 400 × g for 10 min.
5. Pellets were resuspended in red blood cell lysis buffer (2.06 g/l Tris base, 7.49 g/l NH^₄Cl, pH 7.2) for 10 min at room temperature.
6. After resuspending SVF cells in HBSS containing 2% fetal bovine serum (FBS), tissue clumps were allowed to settle for 1 min.
7. Suspended cells were passed through 100-μm and then 40-μm cell sieves. Cell suspensions (15 ml) were applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes.
8. After centrifugation (400 × g, 30 min), cells at the gradient interface were collected, washed in HBSS, and passed through a 30-μm mesh. Cell counts and viability assessment were performed.

Isolation of ADASC from SVF
1. CD45⁺ cells were removed using magnetic beads coupled to mouse antihuman CD45 MAb and a superMACS magnet according to instructions provided by the manufacturer.
2. Magnetic beads were also used to split the remaining CD45^- cells into CD31⁺ and CD31^- cells.
3. CD45^- cells were incubated with FITC-conjugated mouse anti-human CD31 MAb at 1:40 dilution for 15 min at 4°C. After washing, antibody-coated cells were incubated with anti-FITC Microbeads for 15 min at 4°C.
4. Microbead-coated CD31⁺ cells were retained in LS columns, allowing the initial isolation of CD31^- cells, and later flushed out separately according to the manufacturer's protocol.
5. Flow cytometry was used to confirm separation. We never observed more than 8% contaminating cells in either population.

Culture of Freshly Isolated ADASC
1. Immediately after separation, CD31+ and CD31- cell subsets were washed and resuspended in DMEM/F12 containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B.
2. Cells were added to 25-cm² culture flasks (105 cells/flask) and cultured at 37°C in an atmosphere of 5% CO_² in humid air.
3. After 7 d, attached cells were passaged by trypsinization and cultured in DMEM/F12 containing 10% FBS without amphotericin B. Medium was replaced every third day.
4. Generation of clonal cell lines from CD31^- cells was achieved by culturing single fresh cells in separate wells.
5. Using a micropipette, a single cell was placed into each well of 48-well plates containing DMEM/F12 with 20% FBS, antibiotics, and 2.5 μg/ml amphotericin B.
6. Colony-forming ability was assessed after 21 d. At that time, colonies were passaged and cultured further in DMEM/F12 containing 10% FBS without amphotericin B.