Collagenase a type 1

A liposuction aspirate protocol was used to
obtain 200 mL of abdominal adipose tissue under
local anesthesia. The lipoaspirate was washed with
phosphate-buffered saline (PBS) to remove tissue
debris after which 100 mL of PBS that contained 0.1%
w/v collagenase A type I (Sigma, USA) was added
to the isolated tissue, followed by incubation at 37˚C
for 60 minutes. Collagenase activity was neutralized
using MEM medium (Gibco, USA) along with 10%
FBS (Gibco, USA). Cell pellets were resuspended in
culture medium after centrifugation at 2000 rpm for
10 minutes and then transferred to culture flasks for
72 hours at 37˚C and 5% CO₂. The culture medium
in the flasks was changed every 3 days and cells
were passaged 3 times. Lipoaspiration procedure and
cell preparation were carried out in Department of
Regenerative Biomedicine, Royan Institute.

We used the following materials in this clinical
trial study: collagenase A type I (Sigma, USA), fetal
bovine serum (FBS, Gibco, USA), MEM Alpha
1x (Gibco, USA), L-glutamine (Gibco, USA),
antibiotic-antimycotic solution (Gibco, USA),
trypsin-EDTA (Gibco, USA), CD90-fluorescein
isothiocyanate (FITC), CD73-phycoerythrin (PE),
CD105-PE, CD34-FITC, CD45-PE, CD11b-FITC,
CD44-FITC (Sigma-Aldrich, USA), colcemid
solution (Invitrogen, USA), and dimethylsulfoxide
(DMSO, Gibco, USA). One-way ANOVA test was
used in this study which was not significant (P>0.05).