Tissue collection and ADSVF preparation
agitation
STERILE, MINOR, INFILTRATED, SEPARATION
Fat grafts were obtained by liposuction under local anesthesia, in a minor surgical procedures room equipped with necessary items for emergency situations. Briefly, the periumbilical region (9 patients) or posterior thigh (1 patient) was infiltrated with 500 ml of Klein’s formula. Liposuction was performed with a 20-ml syringe and 3-mm diameter blunt cannula with 2-mm openings. A total of 100 ml of fat was harvested, from which 60 ml was transferred to sterile flask containing Hank’s balanced saline solution with 5% albumin and was immediately transported to the laboratory. The adipose tissue was decanted for 10 minutes in a laminar flow cabinet for fraction separation. The upper fraction comprised disrupted adipocytes (oil), and the lower fraction was a fluid portion containing the anesthetic solution, erythrocytes, and dead cells. These both fractions were removed. The middle fraction was washed twice in phosphate-buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and subsequently incubated for 20 minutes in a type II collagenase solution (Sigma, St. Louis, MO, USA) in PBS at a concentration of 2 mg/ml of lipoaspirate. Allow the tubes to incubate at 37°C for 30 minutes with constant agitation. This was followed by addition of ethylenediaminetetraacetic acid (Sigma) in PBS. To separate the ADSVF from the floating adipocytes, this digested tissue was centrifuged at 800 × 