Effects of different liposuction techniques on the isolation of mesenchymal stem cells

Background: In recent years, fat grafts have become useful in plastic surgery. They are mainly used to fill soft tissues, refine breast reconstructions, and for volumetric facial rejuvenation. They are also a rich source of mesenchymal stem cells (i.e., adipose-derived stem cells [ADSCs]), which directly influence fat graft survival. Since ADSCs play an important role in angiogenesis and adipogenic differentiation, it is essential to optimize their isolation. Therefore, in this study, we evaluated and compared 2 procedures used to isolate viable cells from the stromal vascular fraction of abdominal adipose tissue and assess the expressions of surface markers. Methods: We examined 9 female subjects who were scheduled to undergo liposuction. The adipose tissue was isolated from the abdominal infraumbilical region. Fat (20 mL) was collected from the right side by using a cannula attached to a syringe; the plunger was pulled back every 2 cm3 to create low-pressure suction (manual group). The same procedure was repeated on the left side, but the cannula was attached to a sterile and intermediate collector coupled to a vacuum pump that provided a constant negative pressure of 350 mmHg (pump group). The samples were centrifuged, and the adipocytes of the intermediate layer were counted, cultured, and immunophenotyped. Results: The isolation of abdominal adipocytes with a pump providing a negative pressure of 350 mmHg yielded a higher concentration of cells in the stromal vascular fraction than that obtained using 10-mL syringes and low-pressure suction, although the difference was not significant. Conclusions: A negative pressure of 350 mmHg may be safely applied to isolate ADSCs. The cell yield did not indicate any statistically significant difference between the techniques.


INTRODUCTION
Since the advent of fat grafts in 1893, this technique has been used as an important procedure to fill soft tissues 1 .Fat grafts were originally isolated as small or large fragments 1,2 ; subsequently, they could be isolated as smaller fat globules due to the introduction of liposuction 3 .Coleman 4 developed a technique that systematized the isolation, preparation, and transfer of adipose tissue in the recipient bed to improve the graft integration of differentiated adipocytes and provide long-lasting clinical results.
The physiology of fat graft integration involves an initial phase of plasmatic imbibition, angiogenesis, survival of ma ture adipocytes, and adipogenic differentiation of the graft and recipient bed precursors 5 .After the discovery of pre-adipocytes in 1926 1 , several experimental studies de monstrated the ability of these precursors to develop into differentiated and functional adipose tissue [6][7][8] .Zuk et al. 9 isolated cells in the stromal vascular fraction of adipose tis sue that could undergo pluripotent differentiation in a si milar manner to the mesenchymal stem cells of the bone marrow.These cells were termed as adipose-derived stem cells (ADSCs).
The adipose tissue is used as a rich source of mesenchymal stem cells for tissue engineering and regeneration.Therefore, optimizing the procedures for isolating and preparing ADSCs is necessary to achieve the highest number of cells per gram of suctioned adipose tissue.In this study, we evaluate and compare the 2 current procedures used to isolate viable cells from the stromal vascular fraction of abdominal adipose tissue, and assess the expressions of surface markers.

METHODS
We examined 9 healthy female subjects who were scheduled to undergo cosmetic liposuction under general anes-thesia.All subjects signed an informed consent form approved by the Research Ethics Committee of the Institute of Biosciences of the University of São Paulo.
Adipose tissue was harvested from the infraumbilical abdominal region.Twenty milliliters of fat was collected from the right side using a cannula (2.5 mm in diameter and 15 cm in length) coupled to a 10 cm 3 screw-thread type syringe.The plunger of the syringe was pulled back every 2 cm 3 to generate low-pressure suction.This group was called the manual group.
In addition, 20 mL fat was collected from the left side, using a cannula (3 mm in diameter and 25 cm long) coupled to an intermediate sterile collector attached to a pump providing a negative constant pressure of 350 mmHg.This group was called the pump group.
The samples were appropriately labeled and then centrifuged for 3 minutes at 700 × g.Three separate layers were obtained after centrifugation, and the upper and lower layers were discarded.The fat obtained by isolating the intermediate layer in the 2 sample groups was immediately sent to the Laboratory of Genetics of the Institute of Bioscience of the University of São Paulo for analysis.Cells present in the samples were subsequently counted, cultured, and immunophenotyped.
Fat samples were subjected to enzymatic digestion with 0.5 mg/mL collagenase type Ia (Sigma Aldrich) and maintained for 30 minutes in a water bath at 37°C.The more viscous liquid phase was collected, and the collagenase was inactivated in Hank's balanced salt solution (Gibco, Invitrogen).The suspension was centrifuged for 5 minutes at 500 × g to precipitate cells; the supernatant was discarded.
The cell pellets were resuspended, and each sample was divided into 4 aliquots.One aliquot was stained with 0.05% Trypan blue for automated cell counting (Countess Automated Cell Counter, Invitrogen); the final concentration Effects of different liposuction techniques on the isolation of mesenchymal stem cells was determined by counting the cells present in each cubic centimeter of digested fat.Another aliquot was cryopreser ved for further analysis, when required.
Thereafter, adherent cells were washed with buffered sa line solution and detached with trypsin (0.125% trypsin, 0.02% EDTA in PBS).Small aliquots of 5 × 10 3 cells each were prepared and stained with monoclonal IgG antibo dies against CD29, CD 31, CD34, CD44, CD45, CD73, CD90, and CD105 membrane proteins.Flow cytometry was performed with the Guava EasyCyte, and the results were analyzed with the Guava ExpressPlus program (Guava Technologies Hayward).
Paired Student's t-tests were used to compare the num ber of cells obtained in the manual and pump groups.The level of significance was set at P < 0.05.

RESULTS
The number of cells isolated from the fat stromal vascular fraction of the infraumbilical region varied greatly among the 9 female subjects; the average age of the subjects was 46.7 years (range, 22-76 years), and average body mass index (BMI) was 24.9 kg/m 2 (range, 22-29.6 kg/m 2 ) (Table 1).
The average number of cells isolated using the manual and pump techniques were 1.35 × 10 6 and 1.77 × 10 6 cells, respectively.
Although the pump group tended to have more cells than the manual group (Figure 1), the difference was not significant (P = 0.094).
The immunophenotyping performed in P0 cells showed no expression and very low expression of endothelial (CD31-) and hematopoietic lineage markers (CD34+ and CD45+), res pectively (Figure 2).However, the markers present in the mesenchymal cell population (i.e., CD29+, CD44+, CD 73+, CD90+, and CD105+) appear to be highly expressed in cells isolated from the stromal vascular fraction of the abdominal adipose tissue harvested using both techniques (Figure 3).

DISCUSSION
A long-term review of fat grafts reveals the high variability of results obtained with this procedure.However, more satisfactory results were achieved by authors who de veloped procedures aimed to manipulate and harm the least possi ble number of adipocytes during isolation, preparation, and grafting 10,11 .The last phase requires appropria te cellular nu trition and respiration, achieved by plasma tic imbibition, which leads to neoangiogenesis 5 .The graft re-   quires a higher concentration of adipocyte precursors of the stromal vascular fraction because of their crucial role in neoangiogenesis, adipogenic differentiation, and consequent long-term volumetric maintenance of the graft 12,13 .The number of nucleated cells assessed by cell counting was higher in the stromal vascular fraction of the pump group, although the difference was not significant.Although the Trypan blue assay is not considered a direct cell viability assay since it does not take into consideration cellular metabolism, it was used to stain cells of the stromal vascular fraction of the adipose tissue, which include ADSCs, macrophages, pericytes, endothelial and hematopoietic stem cells, pre-adipocytes, and fibroblasts among others.
Immunophenotyping revealed that the negative pressure did not alter the expressions of surface markers at P0.Moreover, the results demonstrate that the ability of adipogenic differentiation remained unchanged.In addition, the expression profile of surface markers in P0 cells was similar to that found in the literature 14 .
Several substances are added to fat grafts to improve adi pocyte integration, including interleukin-8 15 , platelet-ri ch plasma 16 , insulin 17 , and AMD 3100 18 .However, the use of such substances is restricted to experimental biology be cause of the controversial results obtained with their use.The discovery of pluripotent ADSCs 9 and establishment of a simple procedure to isolate these cells led Yoshimura et al. 19 to develop fat grafts enriched with ADSCs to treat fa cial lipodystrophy and hypomastias 20 as well as complications due to silicone breast implants 21 .However, the integration rates of these grafts vary greatly.
Further clarification is required to elucidate the role of precursor adipose cells in the integration and in the nourishment of fat grafts with stromal vascular cells of differentiated adipose tissue.The absence of randomized prospective studies comparing long-term clinical results of fat grafts with and without cell enrichment means that it remains unconfirmed whether both increased ADSC graft concentration and their prior stimulation should be performed to achieve a higher integration rate.However, procedures optimizing the isolation of cells from the stromal vascular fraction are required.
This study supports the findings of Mojallal et al. 22 and demonstrates that manual aspiration with 10 cm 3 syringes and low vacuum pressure lyse fewer mature adipocytes, although fewer cells are isolated from the stromal vascular fraction.Aguena et al. 23 show that isolation of cells from the lower abdomen below the navel yields more nucleated cells in the stromal vascular fraction than that obtained from the flanks.These findings support those of Jurgens et al. 24 .
Although significant individual variation was present, the cell yield obtained per cubic centimeter fat is concordant with that observed in the literature 25 .Moreover, the time required to reach 85% cell confluence was the same in both groups even though the formation of colony-forming units (CFU-F) was not assessed in this study.This observation demonstrates that a negative pressure of 350 mmHg does not alter cell growth (i.e., doubling time).Kurita et al. 26 show that nucleated cells of the stromal vascular fraction of adipose tissue may be centrifuged at relative forces up to 4200 × g.
Concordant with the results published by Mitchell et al. 14 , homogenous immunophenotyping at P0 was observed in both the manual and pump groups.Moreover, antibody tests indicated the presence of surface markers of mesenchymal stem cell lineage, proving their ability to undergo adipogenic differentiation.

CONCLUSIONS
The results of the present study show that a negative pressure of 350 mmHg may be safely applied to isolate ADSCs and that the cell yields between the manual and pump techniques are not significantly different.The possibility of using these cells in surgery to enrich fat grafts immediately after their extraction requires that immunophenotyping should be performed in the fresh stromal vascular fraction.However, further studies should be performed to establish the optimal enrichment rate of fat grafts with ADSCs to improve longter m clinical outcomes.

Figure 1 -
Figure 1 -Cell yield in the manual and pump groups.

Figure 2 -
Figure 2 -Cells isolated from the stromal vascular fraction of adipose tissue were seeded in flasks and cultured in growth medium until confluence.Cells exhibited an elongated shape with several dendritic extensions.

Table 1 -
Characterization of subjects and number of cells in the stromal vascular fraction.

BMI (kg/m 2 ) Number of cells/mL Pump Manual
BMI = body mass index; F = female; M = male.

Maker Percentage of stained cells in the manual group Number of events Percentage of stained cells in the pump group
Figure3-Percentages of stained cells with different markers and numbers of events in both the manual and pump groups.In the above graphs, the magenta lines indicate the control, whereas the blue lines indicate the expressions of surface markers, which was similar in the manual and pump groups.MANUAL, manual group; PUMP, pump group. of different liposuction techniques on the isolation of mesenchymal stem cells