Adipose tissues were collected from the abdomen by aspiration with a suitable needle from donors who signed a consent form. Approximately 50–100 mL of lipoaspirate were collected 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 a Cell Extraction Kit (BioMedFactory, Ho Chi Minh, Vietnam) according to the manufacturer’s instructions. Briefly, adipose tissue was mixed with an enzyme solution containing collagenase at 37°C for 15 min. Then, 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 PBS to remove any residual enzymes, and resuspended in PBS to determine cell quantity and viability using an automatic cell counter (NucleoCounter, Chemometec, Denmark).

SVF samples were cultured in MSCCult medium (BioMedFactory) containing DMEM/F12 supplemented with antibiotic-antimycotic and 10% fetal bovine serum (FBS). 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 medium was added to each flask. After 7 days, the medium was replaced with 12 mL of fresh media. The medium was subsequently replaced every 3 days until the cells reached 70–80% confluence, and then they were subcultured.

Cell markers were analyzed following a previously published protocol. Briefly, cells were washed twice with PBS containing 1% bovine serum albumin (BSA). The cells were then stained with anti-CD14-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD105-FITC, anti-CD90-PE and anti-HLA-DR-FITC 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 DMEM/F12 containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 nM dexamethasone, 0.1 mM indomethacin and 10% FBS (all purchased from Sigma-Aldrich, St Louis, MO, USA). 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 DMEM/F12 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.

To determine the effect of hypoxia on cell proliferation, the ADSC were seeded at a density of 1000 cells/wellin 96-well plates (Costar, Acton, MA, USA) and culturedat 5% or 20% oxygen. On days 2, 4, 6, 8, 10, 12, 14 and 16, the cells were lysed in 200 μL 0.02% sodium dodecyl sulphate (SDS) in DNase-free water, after which the cell number in thesamples was determined using a Pico-GreendsDNAquantitation kit (Invitrogen). The number of cells was calculatedusing a theoretical value of 6.6 pg DNA/cell. The experiment was performed three times, each in quadruplicate.

Similarly, this procedure was repeated in a tri-gas incubator in which the oxygen concentration was controlled to 5%.

Cell cycle analysis was carried out according to the following protocols. Cells from each group were washed twice with PBS and fixed in cold 70% ethanol for at least 3 h at 4°C. Cells were then washed twice with PBS and stained with 1 mL of propidium iodide (PI; 20 μg/mL). RNase A (10 μg/mL) was added to the samples and incubated for 3 h at 4°C. Stained cells were analyzed by flow cytometry using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ, USA).

ADSCs were cultured in E-plates and the eXCELLI-gence system was used to evaluate cell proliferation. The culture medium was replaced with culture medium supplemented with mAbs against VEGF (clone 4.6.1), VEGFR-1 (clone 6.12) or VEGFR-2 (clone IMC1C11). These mAbs were freshly added to the medium at 1 μg per 1 mL of medium. The cell proliferation, doubling time and cell cycle were evaluated similar to in the normal condition (without mAbs).

The expression of VEGFR-1, VEGFR-2 and PDGFRβ was analyzed following protocol. Briefly, cells were washed twice with PBS containing 1% BSA. The cells were then stained with anti-VEGFR-1-FITC, anti-VEGFR-2-FITC or anti-PDGFRβ-PE (all purchased from Abcam). Stained cells were analyzed by a FACS-Calibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses.

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

Similar to previous studies, SVFs from adipose tissue easily attached to the flask surface. After 24 h of incubation at 37°C, some cells attached and exhibited spindle shapes ( Figure 1 ). These cells continuously proliferated and appeared as fibroblast-like cells after 72 h of incubation. After 7 days, the primary culture reached about 70% confluence. The cells were subcultured to a third passage and used in further experiments.

To examine whether these ADSC cells show MSC phenotypes, the MSC marker profile of these cells was assayed. The ADSC candidates strongly expressed markers such as CD44, CD90 and CD105 ( Figure 2 ), while they did not express CD14, CD45 and HLA-DR. Moreover, they successfully differentiated into three kinds of mesoderm cells including adipocytes, which showed positive Red Oil staining, osteoblasts, which showed positive Alizarin Red staining, and chondroblasts, which showed positive Safranin O ( Figure 2 ).

Based on the proliferation curve, we found that after 6 days, the proliferation of ADSCs was different between hypoxia and normoxia. The doubling time significantly decreased in hypoxia-cultured ADSCs compared with normoxia-cultured ADSCs at the 6th day and 10th day. At the 1st day the doubling time of ADSCs in the two groups was not different (43.5 h vs. 40.11 h in hypoxia and normoxia, respectively), however, after 6 days of culture the doubling time under hypoxia decreased to 40.11 h at 1st day to 30.0 h at 6th day, respectively. These values were significantly different from those under normoxia (43.5 h at 1st day and 40.5 at the 6thday). These results were confirmed by a cell cycle assay. After 1st day in culture, the ADSC’s cell cycle hardly changed under the two conditions. However, the differences became clear after 6th day. The proportion of cells in S phase significantly decreased in hypoxia-cultured ADSCs at 6th day compared to 1st day (29.33 ± 3.06% at 6th day compared to 39.01 ± 2.52% at the 1st day), while the change was not significant in normoxia-cultured ADSCs (40.50 ± 0.29% in 6th day compared to 38.88 ± 1.21% in the 1st day). Figure 3

The concentration of VEGF in conditioned medium under hypoxia was significantly higher than that under normoxia. The results showed that the VEGF concentration in conditioned medium under hypoxia increased at 6th day compared with 1st day ( Figure 4 )(from 671 ± 43.14 pg/mL at 1st day to 1223 ± 50.44 pg/mL at 6thday).While this concentration was non-significantly increased in the normoxia group (from 624 ± 42.44 pg/mL at 1st day to at 724.3 ± 33.94 pg/mL 6th day).

Using flow cytometry, VEGFR-1 and VEGFR-2 expression was evaluated at 48 hand 72 h in the hypoxiatreated culture. The results showed that some ADSCs expressed VEGFR-1 and VEGFR-2 at a low level (less than 1%), while more than 95% of ADSCs expressed PDGFRβ ( Figure 5 ).

ADSC proliferation under hypoxia decreased in culture medium supplemented with anti-VEGF or anti-VEGFR mAbs compared with under hypoxia without mAbs. Both doubling time and cell cycle supported this observation. The proliferation rate of ADSCs in culture medium supplemented with anti-VEGF or anti-VEGFR mAbs was significantly decreased compared with that in normal culture medium (without anti-VEGF or anti-VEGFR mAbs) under hypoxia. However, ADSCs in culture medium supplemented with anti-VEGF or anti-VEGFR mAbs still proliferated more rapidly than under normoxia.

As shown in Figure 6 , anti-VEGF or anti-VEGFR mAbs-supplemented medium decreased the growth of ADSCs compared with normal medium (without anti-VEGF or anti-VEGFR mAbs). The doubling time of these ADSCs was non-significantly between hypoxia and normoxia at 6th day (35.67 ± 0.67 hrs in hypoxia and 40.50 ± 0.29 hrs in normoxia). The proportion of cells in S phase under these conditions also was nonsignificantly at 6thday (37.33 ± 2.51% in hypoxia, and 41.33 ± 2.08% in normoxia, espectively).