Comparison of the Viability and Yield of Adipose-Derived Stem Cells (ASCs) from Different Donor Areas

Materials and Methods

Study protocol and donor sites. The human lipoaspirates were prospectively harvested from 40 women that underwent outpatient cosmetic liposuction procedures on a private basis between June 2012 and July 2016. Some participants conferred fat samples from different regions comprising a total of 53 lipoaspirate samples; inner thigh (n=13), outer thigh (n=9), abdomen (n=9), waist (n=16) and inner knee (n=6). Lipoaspirates were harvested using a tulip low-pressure syringe lipoaspiration system. Blunt-tipped liposuction cannulae with a diameter of 3 mm having small holes near the tip were used. All interventions were performed by a single senior plastic surgeon (A.T) and all samples were processed directly. All participants signed a written consent form and agreed beforehand to participate in the study. The study protocol was in accordance with the World Medical Association Declaration of Helsinki and an approval from our Institutional Review Board was obtained.

Isolation of the SVF and assessment of viability. After harvesting, lipoaspirate samples (Mean volumes of samples are presented on Table I) were directly washed with phosphate-buffered saline (PBS) and then centrifuged at 400 ×g for 15 min. Following removal of the oil, the lipid phase of the lipoaspirate from the top of the tube was obtained and diluted with an equal volume of collagenase digestion solution (Collagenase NB 4G proved grade; SERVA Electrophoresis, Heidelberg, Germany). After 25 min of incubation, an equal volume of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum was added to stop enzymatic digestion. The supernatant, containing adipocytes, was separated from the pellet, containing the stromal vascular fraction, with centrifugation. The isolated stromal vascular fraction was filtered through a 100-μm nylon filter and then processed for density gradient by centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Thereafter, the mononuclear cell layer was carefully aspirated and the total number of stromal vascular fraction cells was counted. Viability of SVF was evaluated using the Trypan blue exclusion assay. Cell suspension (10 μl) was diluted 1:1 with 0.4% Trypan Blue solution (Thermo Fischer Scientific, CA, USA). Cells were counted using a hemocytometer under light microscopy and the percentage of live cells was calculated. All activities were performed under good manufacturing practice (GMP) standards.

Isolation of ASCs through SVF culture. Isolation of ASCs was achieved through stromal vascular fraction culture. In brief, 1×10⁷ SVF cells and 20 ml of pre-warmed Non-hematopoietic Expansion Medium (Miltenyi Biotec, Auburn, CA, USA) containing 1% of penicillin-streptomycin (Miltenyi Biotec, Auburn, CA, USA) were added to a 75-cm² cell culture flask and cultivated at 37°C, 5% CO₂, and 95% humidity. Following one day, non-adherent cells were removed by washing the flask with PBS. The adherent ASCs were cultured with the addition of fresh prewarmed (37°C) Nonhematopoietic Expansion Medium (20 ml). The culture medium was replaced every two days and passaging was performed every 5 days. Briefly, cells were washed with PBS and detached after incubation with 0.5 mM EDTA/0.05% trypsin (Invitrogen, Carlsbad, CA, USA) at 37°C for 15 min. Then, ASCs were washed with fresh Nonhematopoietic Expansion Medium and transferred to a new T-25 flask (1.25×10⁵) with 5 ml of fresh Nonhematopoietic Expansion Medium. Finally, cell number was counted on each passage to calculate cell doubling time.

Detection of ASCs and assessment of viability. To characterize ASCs subpopulations from the fresh lipoaspirate samples, phenotype of cultured ASCs was examined after labeling with a panel of monoclonal antibodies against mesenchymal and hematopoietic stem cell markers (BD Biosciences, San Jose, California, USA) including stem cell markers (i.e. CD90 and CD105) and hematopoietic markers (i.e. CD45 and CD34). Briefly, 1×10⁵ SVF cells were stained with 0.05% CD90-PE-cy (PE-Cy™7 Mouse Anti-Human CD90 Clone 5E10, BD Biosciences, USA), 0.05% CD105-PerCP (PerCP-Cy™5.5 Mouse anti-Human CD105 (Endoglin) Clone 266, BD Biosciences, USA), 0.2% CD34-APC (APC Mouse Anti-Human CD34 Clone 581, BD Biosciences, USA), 0.2% CD45-PE (PE Mouse Anti-Human CD45 Clone HI30, BD Biosciences, USA) and 0.05 CD73-PE-cy (PE-Cy™7 Mouse Anti-Human CD73 Clone AD2, BD Biosciences, USA). After reacting with antibodies at 4°C for 30 min, the cells were washed with PBS and analyzed by flow cytometry using BD FACSDiva software v6.1.3 (Becton Dickinson, San Jose, CA, USA). Viability of ASCs was assessed using the 7-Aminoactinomycin D (7-AAD) staining (Thermo Fisher Scientific, Waltham, MA, USA) with a flow cytometer.

Statistical analysis. Numerical data are presented as mean±standard deviation. Qualitative variables are presented as absolute and relative frequencies. All measurements were compared by using analysis of variance (ANOVA) followed by t-test. Statistical analysis was performed with the IBM SPSS v.22 statistical package. A value of p≤0.05 was considered significant and all p-values were two-sided.