Protocol for obtaining and characterizing the stromal vascular fraction of human adipose tissue by mechanical dissociation: an original article

The present technical study presented the mechanical dissociation protocol of adipose tissue from manual liposuction, as well as showed the immunobiological characterization (immunophenotyping), cell viability of the stromal vascular fraction of human adipose tissue, and cell concentration per milliliter. It was observed that the technique of the present study obtained a characterization yield of the stromal vascular fraction of the adipose tissue in terms of cell viability, cell concentration per milliliter, and immunophenotyping comparable and superior to most published studies with good scientific evidence.


Introduction
In the regenerative medicine scenario, the identification of cells from the stromal vascular fraction (SVF) of human adipose tissue has opened a promising new field [1,2]. After decades of dealing with liposuction and fat grafting, it is possible to harvest adipose tissue in almost any desirable volume, quickly and easily and with relatively low morbidity, representing an unprecedented source of adipose-derived stem cell (ADSC) [3][4][5][6][7][8][9][10].
In this sense, cell-based therapies using liposuction are gaining popularity due to their regenerative potential [1,2,11]. Various liposuction processing devices/systems have become available to isolate cells for therapeutic use, with published evidence reporting their clinical relevance. However, few studies have parallel analyzed the composition of their minimally manipulated cellular products, vital information to understand the mechanisms by which these therapies can be effective [12].
In this context, the practical applications of cell therapy are still limited, but this new perspective of potential applications introduces a concern with the quality of disposable fat as a source of donor cells and liposuction procedures must be properly used and must be optimized in this sense. Liposuction is the method of choice for ADSC extraction, with variations in protocols using all the fat obtained or even just the saline/blood fraction of liposuctions, but tissue harvesting procedures are often underestimated as a factor to impact results. The laboratory protocols for extracting ADSC from the material obtained should also be duly evaluated, from simple cell counts to its characterization as mesenchymal tissue and its biological properties in vitro [11,12].
Therefore, the present technical study presented the mechanical dissociation protocol of adipose tissue from manual liposuction, as well as showed the immunobiological characterization (immunophenotyping), cell viability of the stromal vascular fraction of human adipose tissue, and cell concentration per milliliter.

Mechanical Dissociation Protocol
After the liposuction process in the operating room, 30.0 mL of adipose tissue was voluntarily donated by the patient for the characterization of the present DOI: 10.54448/mdnt22412

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MedNEXT Journal of Medical and Health Sciences research protocol and was placed in sterile 15 mL falcon tubes for sample homogenization. 10 samples were established from the 30 mL of liposuction. After that, the adipose tissue was aspirated in a 3 mL needleless syringe. The adipose tissue was subjected to mechanical dissociation using two syringes connected to pass the sample in both directions until obtaining a highly liquefied sample, with the disaggregation and separation of cells from the stromal vascular fraction (cellular solution). The solution containing the dissociated cells was placed in syringes that remained in an upright position for ten minutes. Then, the cell solution was centrifuged at 800xg to separate cell debris and mature adipocytes. The collected material was again centrifuged for 10 minutes at 800xg. The precipitate (pellet) obtained was resuspended in 1.0 mL of sterile hospital saline solution, and 10 µL was removed for counting and determination of cell viability and flow cytometry analysis (immunophenotyping).

Cell count and viability
Trypan blue is a "vital dye", that is, it crosses the membrane of dead cells, but not living cells. Thus, by resuspending the cells in a trypan blue solution, it is possible to assess the viability of the suspension. Counting the number of cells in a suspension, using the Neubauer chamber, is based on counting the number of cells in a sample of a defined volume, which is then converted into the number of cells per mL or the total number of cells in the sample, with the appropriate formulas. According to Dominici et al. 2006 [8], 10.0 uL of cell solution was placed in the Neubauer chamber for quantification of live and dead cells, with replicates of n=3.0. The Neubauer chamber has its central strip in a millimetrically determined position below the sides, so that the placement of a coverslip, well adhered to the edges of the depression, originates a chamber with a well-determined volume. After placing a sample of the cell suspension in this chamber, so that it is filled, but without extravasation, the viable (unstained) cells are counted under the microscope. The chamber has four quadrants (Q1, Q2, Q3, and Q4) with an area of 0.1mm² each. By integrating a glass coverslip into the Neubauer Chamber, a height of 0.1 mm is obtained.

Immunophenotyping Assay
After removing the stromal vascular fraction from the falcon tubes, they were suspended in 1.0 mL of PBS supplemented with 3.0% human albumin (Human Albumin Grifols® 20.0%, PBS/A). The resulting suspension was subjected to the determination of the number and proportion of non-viable cells by the Trypan blue staining method, followed by adjustment of the viable cell density to 106 cells mL -1 . The immunophenotypic evaluation of these cells was performed using the immunophenotyping technique by flow cytometry in a FACSCanto II® cytometer (BD Biosciences), using monoclonal antibodies conjugated with fluorochromes, and their respective isotypic controls. Fresh FEV cells homogenized in PBS/A were aliquoted in 100.0 µL and dispensed into 5.0 mL tubes for flow cytometry. Afterward, 5.0 μL of each antibody was added to the tubes and homogenized by vortexing. The suspensions were then incubated for 15.0 min at room temperature, in the dark. Then, 400.0 μL of PBS/A was added to each tube, followed by homogenization by shaking and centrifugation at 400xg for 10.0 min. The supernatant was discarded and the pellet was homogenized in 500.0 μL of PBS/A. To tube 3 of each sample, 5.0 μL of 7-AAD was added moments before acquisition in the cytometer. The CTMA was then acquired in a cytometer coupled to the analysis software BD FACSDiva II® (BD Biosciences) and subsequently analyzed using the Infinicyt® software (Cytognos) for the binding/marking of each antibody (marker expression) and 7-AAD (non-viable cells) [8].

Ethical Approval
The present study was approved by the Research Ethics Committee of the Hospital Beneficência Portuguesa de São José do Rio Preto/SP, under approval process number 030427/2015. The term of consent was applied before the analysis of in vitro cell samples from adipose tissue. Table 1 presents the results of cell concentration (cells/mL) and cell viability (%) of the 10 samples obtained from the stromal vascular fraction of adipose tissue through mechanical dissociation. Analyzes were performed immediately after liposuction collection. Table  2 presents the results of immunophenotyping of the stromal vascular fraction of a representative sample of ten samples. The CD44+ surface marker is highly specific for adipose mesenchymal stem cells. Analyzes were performed immediately after liposuction was collected through mechanical dissociation.

Vol 3 Iss 4 Year 2022
MedNEXT Journal of Medical and Health Sciences

Vol 3 Iss 4 Year 2022
MedNEXT Journal of Medical and Health Sciences Source: Own authorship.
The present study presented results comparable to 15 studies that were published on techniques of mechanical dissociation of adipose tissue to obtain the stromal vascular fraction. The 15 studies were analyzed and published by the authors Liu et al. 2022 through a systematic review [12]. All studies reported the cell concentration (number of cells regardless of phenotype per milliliter of liposuction) for their devices/systems (range 0.005-21×106) [12]. The present study was able to obtain an average of 1.3 ± 0.2 x107 cells/mL, that is, about one thousand more cells per mL. Also in the study by Liu et al. 2022, ten articles reported cell viability (the measure of live cells ranging from 60 to 98%) [12]. In the present study, cell viability was 99.5± 0.3%, which is higher than the average of published articles. Furthermore, in the study by Liu et al. 2022, 11 articles performed immunophenotypic analyzes of cell subtypes [12]. In the present study, a yield of 75.1 ±12.6% was obtained for the CD44+ cell surface marker, which is highly specific for mesenchymal stem cells.

Conclusion
It was concluded that the technique of the present study obtained a characterization yield of the stromal vascular fraction of adipose tissue in terms of cell viability, cell concentration per milliliter, and immunophenotyping comparable and superior to most published studies with good scientific evidence.

Vol 3 Iss 4 Year 2022
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