SVF Isolation Methods and Wound Healing

In-Vitro Comparative Examination of the Effect of Stromal Vascular Fraction Isolated by Mechanical and Enzymatic Methods on Wound Healing

Abstract

Background

Stem cells derived from various sources, including adipose tissue, bone marrow, peripheral blood, cord blood, and amnion, are known to enhance wound repair of damaged tissues, and therefore provide a useful resource for chronic wound treatment. In fact, recent studies have demonstrated that adipose-derived stem cells (ADSCs) from in-vivo animal tests showed human dermal fibroblast proliferation by both direct cell-to-cell contact and secretory-induced paracrine activation, thereby significantly reducing wound size, accelerating re-epithelialization, and thus shortening the time required for wound closure.

The use of a heterogeneous cell population of adipose-derived stromal vascular fraction (SVF) is known to be one of the most promising therapeutic strategies for wound healing based on certain physiologic characteristics such as epithelialization, angiogenesis, and immunomodulation.

Although adipose tissue consists mostly of adipocytes, it is an important mesenchymal stem cell reservoir. SVF is a heterogeneous cell cocktail that is obtained by the destruction of adipose tissue through surgery means. SVF contains stem cells, mesenchymal cells, preadipocytes, endothelial cells, hematopoietic cells, fibroblasts, and pericytes. The use of SVF is a recent development with important clinical implications in plastic surgery and other surgical branches.

The traditional isolation method for obtaining SVF is through enzymatic digestion of human lipoaspirate, which yields E-SVF. Approximately 100,000 to 1,300,000 nucleated cells per gram of lipoaspirate can be obtained by enzymatic digestion with more than 80% cell viability. Enzymatic digestion effectively disrupts the functional extracellular matrix, leaving SVF as a heterogeneous mixture of “naked” ADSCs, pericytes, macrophages, fibroblasts, vascular endothelial progenitors, and blood cells. However, enzymatic digestion of adipose tissue is considered to involve more than minimal manipulation of tissues and has therefore not been approved by the Food and Drug Administration and other regulatory authorities. Furthermore, this isolation method remains expensive, requires “good manufacturing practice” facilities, has long isolation times (90-120 minutes), and raises legal and administrative concerns. In spite of these disadvantages, automated devices used in the isolation of SVF for clinical and surgical applications have spread worldwide. The main advantages of automated devices include a closed environment, minimal risk of contamination, standardization in clinical practice, and the ability to perform the application in operating rooms or health clinics. However, the widespread use of these devices is limited due to their high costs and the requirement to be trained to operate them.

For these reasons, research into mechanically dissociated stromal vascular fraction (M-SVF) was initiated by a number of groups within the regenerative medicine community. Various mechanical isolation methods based on shaking, vibration, and centrifugation were tested and evaluated against enzymatic protocols. Although low yields of SVF cells in terms of cell count and viability were initially reported, subsequent studies have shown that phenotypic characterization of M-SVF is higher than that of E-SVF. The literature also demonstrated that M-SVF contains an existing amount of adipose-derived stromal cells and endothelial cells, which supports its use in regenerative medicine. To further support these findings of higher phenotypic characterization in M-SVF, the aim of the present study was to investigate the effect of M-SVF and E-SVF by studying the effect of fat tissue on wound healing in vitro.

Methods

This study was started in September 2019 and concluded 5 weeks later in October 2019. It was not necessary to obtain approval from an institutional review board as the SVF isolation was performed in vitro and followed standard protocols. The Declaration of Helsinki was used as a guiding principle for this study. Ten consecutive, nonobese, female patients were preoperatively informed about all surgical procedures, anesthesia, intraoperative video recording, and photography, and gave written consent.

Cell Isolation Methods

A total of 40 mL of lipoaspirate per patient was obtained with a 2-mm-diameter multihole cannula. After decanting, the adipose tissue was partitioned into 2 equal parts for mechanical and enzymatic SVF isolation.

Enzymatic Isolation of SVF

A 20-mL portion of adipose tissue was shaken at a constant speed for 30 minutes in a 37 °C water bath with GMP-grade collagenase NB6 (Serva Electrophoresis, Heidelberg, Germany) at a concentration of 0.1 U/mL and a ratio of 1:1 (w/w). After shaking, the adipose tissue was washed with phosphate-buffered saline and centrifuged at 300 × g for 5 minutes. When centrifugation was completed, the supernatant, which contains fat cells and washing/enzyme liquid, was discarded. The pellet containing SVF cells was then homogenized by adding phosphate-buffered saline in a 1:3 volume ratio. After the cells had been collected by homogenization of cell suspension, they were filtered with a 70-μm sterile filter (LSR00159, BD Biosciences, San Jose, CA, USA).

Mechanical Isolation of SVF

A 20-mL portion of adipose tissue was transferred to 20-mL Luer-lock jointed syringes with the help of a 3-way stopcock (Lipocube SVF Kit; Lipocube, London, UK). Mechanical isolation was achieved in a cube-shaped apparatus with 3 different blade grids at 3 separate Luer-lock ports with a rotating hub path. One syringe injector full of adipose tissue was placed into the first port, while an empty syringe injector was placed into the second port. Between these ports is a filter with 1000-μm holes that reduces the adipose tissue into smaller parcel sizes. This process is repeated with a 750-μm filter between the second and third ports, and a 500-μm filter located between the third and fourth ports, further mincing the adipose tissue into smaller parcels of fat. To remove contaminating erythrocytes, a 1:3 calcium-magnesium balanced buffer solution was added to the adipose product and incubated for 10 minutes at room temperature. Prior to centrifugation, ordinary pistons of 20-mL Luer-lock syringes were replaced with custom-made disarmable pistons with concave, cell-adhesive gaskets. The pistons were detached and the syringes containing the dissociated lipoaspirate were centrifuged at 2000 × g for 10 minutes with the Luer-lock tips directed inward so that the SVF could be collected in the concave gaskets. After centrifugation, the piston tops that had been removed were reinstalled and the fat tissue and buffer solution outside the cell pellet was transferred to an empty syringe with the aid of a 3-way stopcock. As a final step, the cell pellet was homogenized with a saline solution.

Cell Count and Viability

Total nucleated cell count and the viability of each cell cocktail isolated enzymatically and mechanically were measured by flow cytometry (Muse Cell Analyzer; Merck Millipore, Germany) following red blood cell lysis.

Flow Cytometry

Flow cytometry was used to quantify and compare adipose-derived stem phenotypes with using specific clustered of differentiation markers (CD surface markers) and SVF stem potential in all groups. Characterization of ADSCs (CD45–/CD90+/CD73+), endothelial cells (CD90+/CD44+), and pericytes (CD105+/CD73+) was performed. Staining was done with 5 mL of monoclonal antibodies (BD Biosciences, Le Pont de Claix, France). The binding efficiency of the surface markers CD73, CD90, CD105, and CD44 was also examined.

Cell Proliferation Test

The SVF cell populations isolated from each protocol were then grown under the same cell culture conditions. For the cell proliferation analysis, cells obtained by the 2 different methods were plated in 96-well cell culture plates at a concentration of 3 × 10³ cells/hole and treated with low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco Thermo Fisher Scientific, Loughborough, UK) and 1% penicillin-streptomycin-amphotericin mixture for 24, 48, and 72 hours. Cell proliferation was evaluated with the WST-1 test (BioVision, Milpitas, CA), as discussed in the literature. The absorbance value of WST-1 was measured at 540 nm in a spectrometer. The experiment was completed by 3 biological replicates. ATCC adipose-derived mesenchymal stem cells (American Type Cell Culture, Manassas, VA) were used as a control group in order to compare stem phenotypic characterization in the SVF samples. Only 1 cell batch was used throughout the study (adipose-derived mesenchymal stem cells; normal, human, ATCC PCS-500-011).

Wound-Healing Scratch Assay

The two SVF cell populations were plated on 6-well cell culture plates (TPP, Trasadingen, Switzerland) at a concentration of 1 × 10⁶ cell/hole. Allantoin (50 μg/mL), a plant-derived commercialized drug, was used as a positive control to increase migration. ATCC adipose-derived mesenchymal stem cells served as a negative control group without any treatment, and 2% fetal bovine serum (FBS) was used to decrease the migration proliferation potential. Each group was fed with fresh medium containing 2% FBS. Once cells have been incubated and have achieved full surface coverage, a slit model was created in the middle of the container under sterile conditions with the aid of a 200-µL pipette tip. After 48 hours, the closure speed and spacing of this slit was measured by light microscopy with an inverted microscope (Nikon Eclipse TE200; Nikon, Tokyo, Japan) and wound closure analysis was performed with ImageJ software according to the formula: percentage wound closure = ((measurement at 0 h – measurement at 48 h)/measurement at 0 h) ×100.

Gene Expression Analysis with Real-Time Polymerase Chain Reaction

Collagen type I primer, which plays an active role in wound healing, was designed with Primary-BLAST software from the National Biotechnology Center (Bethesda, MD). Total RNA isolation from SVF cell populations obtained by the 2 different methods was isolated with a Total RNA Purification Kit (Norgen, Ontario, Canada) according to the manufacturer’s protocol. A QuantiTect Reverse Transcription kit (Qiagen, Les Ulis, France) was used to transform RNA into cDNA. The mRNA expression level of collagen type 1 gene (Col1A) was analyzed with a QuantiTect SYBR Green polymerase chain reaction (PCR) kit (Qiagen, Les Ulis, France). Reactions were read with an iCycler real-time (RT) PCR system (Bio-Rad, Hercules, CA) according to the manufacturer’s protocols. In the analysis, 18S rRNA was used as a reference gene. ATCC ADSCs served as a control group.

Statistical Analysis

One-way analysis of variance and GraphPad Prism 5 (GraphPad, La Jolla, CA) software were used for statistical analysis with repeated samples. P ≤ 0.05 values were considered as statistically significant. Because the analysis of variance was used to compare cell proliferation and gene expression between the 2 groups (E-SVF vs M-SVF), a post-hoc test was not required. Normal distribution was used. Nonparametric tests were not applied.

Results

Patient Characteristics

After providing written consent, 10 consecutive, nonobese women, with an average age of 39 years (range, 26-52 years) and an average body mass index of 26 kg/m²(range, 17-35 kg/m²) received liposuction to the lateral hip region.

Cell Count and Cell Viability Analysis

The total number of nucleated E-SVF cells isolated was 1.74 × 10⁶ whereas the total number of nucleated M-SVF cells isolated was 0.94 × 10⁶. Cell viability after isolation was 96.67% in the E-SVF group and 97.55% in the M-SVF group. There was no significant difference between the cell viabilities achieved with each SVF isolation method (Table 1, Figure 1).