All patients enrolled in this study were required to sign a consent form. All procedures used in the study were approved by the ethical committee of the hospital. Five adipose tissue samples were used in this study. Fat tissue was collected from the belly by aspiration with a suitable needle. For this, approximately 50–100 mL of lipoaspirate was collected from each patient in two 50 mL sterile syringes. The syringes were stored in a sterile box at 2–8°C and were immediately transferred to the laboratory.

The SVF was isolated from adipose tissues using an ADSC Extraction Kit (GeneWorld, Ho Chi Minh City, Vietnam) according to the manufacturer’s instructions. Briefly, 50–100 mL of lipoaspirate was placed in a sterile disposable 250 mL conical centrifuge tube (Corning, Tewksbury, MA) and washed in Washing Buffer 1 by centrifugation at 1000 g for 5 min at room temperature. Next, the adipose tissue was washed with Washing Buffer 2 as per the procedure for Washing Buffer 1. Then the adipose tissue was digested using SuperExtract Solution containing collagenase at 37°C for 45 min with agitation at 5 min intervals. The cell suspension obtained was centrifuged at 3000 g for 10 min, and the SVF was obtained as the pellet. The pellet was washed with Washing Buffer 3 to remove any residual enzymes, and resuspended in PBS for the determination of cell quantity and viability using an automatic cell counter (NucleoCounter; Chemometec, Denmark).

Activated PRP was derived from the peripheral blood of the same patient as the adipose tissue using a New-PRP Pro Kit (GeneWorld, Ho Chi Minh City, Vietnam) according to the manufacturer’s guidelines. Briefly, 20 mL of peripheral blood was collected into three vacuum tubes and centrifuged at 800 g for 10 min. The plasma was collected and centrifuged at 1000 g for 5 min to obtain a platelet pellet. Plasma in the upper layer was then removed, leaving 3 mL of plasma at the bottom of tube. Finally, PRP was activated using activating tubes containing 100 μL of 20% CaCl2.

Five SVF samples were used for primary culture. SVF samples were cultured in MSCCult medium that contained DMEM/F12 supplemented with antibioticantimycotic, EGF, basic fibroblast growth factor (bFGF) with 10% aPRP or 10% FBS to act as the control. This chosen concentration was based on the findings of a previous publication. The cells were plated at 5 × 10⁴ cells/mL in T-75 flasks (Corning) and incubated at 37°C with 5% CO2. After 3 days of incubation, 6 mL of fresh media were added to each flask. After 7 days, the media were replaced with 12 mL fresh media. The media was subsequently replaced every 3 days until the cells reached 70–80% confluence, where they were subcultured.

The proliferation rate of SVF secondary cells was evaluated by the eXCELLIgence system (Roche Applied Science, Indianapolis, IN, USA). Specifically, cells were seeded at 1 × 10³ cells/well into a 96-well Eplate in triplicate. The culture plates were placed into the eXCELLIgence system and incubated at 37°C with 5% CO2. This chosen concentration was like the previous result. In contrast to the primary culture medium, the secondary culture medium was MSCCult supplemented with 5% aPRP. The cell doubling time and slope were calculated using the software associated with the eXCELLIgence system.

Cell markers were analyzed following a previously published protocol. Briefly, cells were washed twice in PBS containing 1% bovine serum albumin. The cells were then stained with anti-CD14-FITC, anti-CD34- FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD73-FITC, anti-CD90-PE antibodies (all purchased from BD Biosciences, San Jose, CA, USA). Stained cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses.

For differentiation into adipogenic cells, ADSCs were treated as described previously. Briefly, cells were plated at 1 × 10⁴ cells/well in 24-well plates. At 70% confluence, the cells were cultured for 21 days in IMDM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 nM dexamethasone, 0.1 mM indomethacin, and 10% FBS (all purchased from Sigma- Aldrich). Adipogenic differentiation was evaluated by observing lipid droplets in cells under a microscope.

For differentiation into osteogenic cells, ADSCs were plated at 1 × 10⁴ cells/well in 24-well plates. At 70% confluence, the cells were cultured for 21 days in IMDM containing 10% FBS, 1 × 10^-7 M dexamethasone, 50 μM ascorbic acid-2 phosphate, and 10 mM β- glycerol phosphate (all purchased from Sigma Aldrich). Osteogenic differentiation was confirmed by Alizarin red staining.

Secondary cells at the 15th passage were treated with 0.10μg/ml colcemid for 24 hours. Cells were harvested by trypsinization, and subsequently incubated in a hypotonic solution for 1 h at 37°C. The cells were then fixed at least 3 times in Carnoy’s solution, which included an overnight fixative step. The fixed cell suspension was placed onto prepared slides, and dried at 60°C for 3 h. These lames were used for G-banding staining. Chromosomes were visualized using Ikaros software (MetaSystems, Altlussheim, Germany) in the upright microscope (A2 Image, Carzeiss, Germany).

The tumorigenicity of ADSCs at third and tenth passage was examined in athymic nude mice. All manipulations with the mice were approved by the Local Ethics Committee. Each mouse was injected subcutaneously with 5 × 10⁶ ADSCs (three mice per group). As a positive control, mice were also injected with breast cancer cells at a different site. Tumor formation in mice was followed for one month.

Significant differences between mean values were assessed by t-tests and analysis of variance (ANOVA). A P-value of <0.05 was considered statistically significant. All data were analyzed by Prism 6 software.

ADSCs cultured in MSCCult supplemented with either FBS or PRP adhered to the flask surface after 3 days, and exhibited a stromal phenotype that was similar to fibroblasts. These cells continuously proliferated to reach 70–80% confluence after 10 days when cultured in the MSCCult medium supplemented with PRP ( Figure 1 ), after 15 days when cultured in the MSCCult medium supplemented with FBS. These cells were sub-cultured and passaged five times, after which they were used to determine the presence of MSC surface markers. The results are presented in Figure 2 Cells cultured in both media exhibited several MSC markers, which included CD44 (99.12%), CD73 (99.81%), CD90 (95.08%) while other markers were not prevalent in the ADSC populations, including CD14 (1.35%), CD34 (1.02%), and CD45 (1.32%) ( Figure 2 ).

ADSCs successfully differentiated into three different mesoderm layers, including adipocytes, and osteoblasts. In the adipogenesis medium, ADSCs in both PRP-medium and FBS-medium groups started to accumulate lipid droplets in the cytoplasm by day 14. These lipid droplets gradually increased in size and were clearly recognized in an inverted microscope at day 21. These drops were confirmed as lipid when they were positive with Oil Red dye ( Figure 3 ). In the osteogenesis medium, ADSCs rapidly changed their shape to become similar in morphology to osteoblasts, and enhanced their matrix protein production. These cells were positive for Alizarin red ( Figure 3 ).

ADSCs rapidly proliferated in the FBS free medium compared to FBS medium

As shown in the Figure 4A , ADSCs in the PRP-supplemented medium significantly increased compared to in FBS-supplemented medium. After early 24 h, the proliferation curve was non-significantly different in both groups. However, ADSCs in medium supplemented with PRP began to proliferate faster after 24 h when compared to those cells grown with FBS supplementation. The differences in cell proliferation between the two groups were more distinct after 72 h. These results were confirmed in doubling time ( Figure 4B ) and slope values ( Figure 4C ) analysis. The doubling time of ADSCs supplemented with PRP was significantly reduced as compared to that of those cells supplemented supplemented with FBS. Conversely, the slope value for those cells supplemented with PRP was significantly higher than those cells treated with media plus FBS.

ADSCs maintained the karyotypes after 15th passages

ADSCs cultured in PRP-supplemented medium at 15th passage were used to analyze karyotypes. The results showed that cells at 15th passages exhibited normal karyotypes (2n = 46) ( Figure 5 ).

ADSCs could not cause tumors in the mouse

Cultured ADSCs at 15th passage were injected to NOD/SCID mice to determine their tumorigenicity. The results indicated that these ADSCs do not cause tumors at high doses (10⁶ –10⁷ cells). While injection of an equal amount of breast cancer cells formed tumors in the NOD/SCID mice. These results were similar to ADSCs cultured in the FBS-supplemented medium.