Flow chart of the methodology.
SEPARATION, INFILTRATION
(A) Fat grafts are obtained by abdominal liposuction. (B) A 60 ml volume was sent to the laboratory. (C) The enzymatic digestion of fat was performed by a collagenase separation method. (D) The initial adipose-derived stromal vascular fraction (ADSVF) was divided into two samples. (E) The first was a 1 ml sample for the characterization and control of the cell culture. (F) The second was a 2 ml sample that is reserved for administration to the patient in a lipoaspirate mixture by infiltration into the hand. (G) The differentiation process.To verify that the ADSVF contained stromal cells, they were cultured and stimulated for their differentiation into mature adipocytes. The cells were seeded in 6-well culture plates in Dulbecco’s modified Eagle’s medium and were supplemented with fetal bovine serum and antibiotic/antifungal agents. Upon reaching 90% confluence, the cells were differentiated into mature adipocytes using a differentiation cocktail (2.5 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μg/ml insulin). As a control, a plate containing cells in the culture medium was left without adipogenic stimulation. After 20 days of differentiation, the cells were stained with red oil to assess fat accumulation (mature adipocyte phenotype), and the fat content in each culture was quantified by spectrophotometry (