Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.
The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.
Over the past few years, regenerative plastic surgery has emerged as an additional pillar of plastic surgery1. Regenerative plastic surgery aims to restore damaged tissue by transferring soluble factors, cells, and tissue harvested from the patient to promote tissue restoration in a minimally invasive manner2. Adipose-derived stem cells (ASCs) have gained attention due to their ability to differentiate into multiple mesenchymal lineages, making them a promising candidate for regenerative medicine research3. Their cytokine profile displays angiogenic, immunosuppressive, and antioxidative effects4.
Traditionally, ASCs were isolated from adipose tissue using an enzymatic approach with collagenase, resulting in a stromal vascular fraction (SVF), which was subsequently cultured to obtain ASCs. These laboratory-based technologies are costly, time-consuming, and importantly, subject to strict regulatory restrictions, complicating clinical translation5,6,7. In contrast, mechanically isolated stromal vascular fraction (mSVF) protocols offer the clinical benefits of not only bypassing regulatory issues but also minimizing contamination risks8,9.
Numerous protocols to mechanically isolate the SVF have been described10. Amongst these, the shifting protocol published by Tonnard et al. has gained the most attention amongst regenerative surgeons11. The fat collected through standard liposuction procedures, known as lipoaspirates, can be transferred between two handheld syringes attached to a connecting device, resulting in a liquid form referred to as nanofat. The obvious benefits of these mSVF isolation protocols include reduced processing time, minimal risk of contamination, as the whole procedure is done in a well-controlled environment, and possible immediate clinical translation12.
Preclinical and clinical evidence indicates that the properties of mSVF, including cell viability and wound healing properties, are comparable to standard enzymatic isolation methods12. The potential of mSVF in promoting wound healing in rat and murine models was validated through in vivo studies by Chen et al. and Sun et al.13,14. However, there is a lack of available data regarding wound healing in the clinical setting. Promising results were reported when a study group performed autologous fat transplantation in an 83-year-old patient who had a wound with an exposed implant in an open fracture of the lower extremity15. Furthermore, Lu et al. conducted a comparison between mSVF and negative pressure wound therapy in a cohort of 20 patients with chronic wounds16. Their findings revealed that mSVF treatment resulted in a higher rate of wound healing compared to negative pressure wound therapy16. Both mentioned studies injected mSVF alone or in combination with a gel into the targeted wound area15,16.
In the real-world scenario, clinical application of mSVF is limited due to unpredictable absorption rates at recipient sites17,18. Scaffolds promise a remedy to this issue, as they assist in cell retainment, vascularization, and integration into the surrounding tissue19,20,21. Fibrin hydrogels are a commonly used, FDA-approved tool used in surgical disciplines and have been shown to be an effective carrier of mSVF19. Fibrin gel is a biopolymeric material which provides several advantages in functioning as a cell carrier: it displays excellent biocompatibility, promotes cell attachment, and is capable of degrading in a controllable manner22,24,25. Additionally, it demonstrates minimal inflammatory and foreign body reaction and is easily absorbed during the natural course of wound healing22. We believe that the diverse regenerative capabilities of mSVF cells mentioned and the advantageous combination with a fibrin hydrogel can provide an innovative approach to enhance wound healing processes. Overall, this approach allows for an efficient topical delivery of viable mSVF cells. We hereby present the protocol that combines mSVF with a fibrin hydrogel intended for application in wound healing purposes.
This study was performed in accordance with the Declaration of Helsinki. All adult donors provided written informed consent to allow further use of the collected tissue samples. The protocol follows the guidelines of our institution's human research ethics committee.
1. Harvest of adipose tissue
2. mSVF-Isolation
3. Manufacturing of mSVF-fibrin hydrogel
4. Viability assay and histology
Resazurin assay
We first examined the in vitro cell viability of the mSVF cells. For this purpose, we conducted a resazurin cell viability assay on days 0, 3, and 7. The cell viability at days 0, 3, and 7 of a total of four samples are shown in Figure 1. The values of day 0 serve as the baseline and were set as 100%. At day 3, the positive control (mSVF) showed a slight decrease to 78.92% (± 5.33%), while the mSVF-fibrin hydrogel combination remained at 96.48% (±1.89%). Similar trends were observed at day 7, where mSVF measured 77.46% (±3.90%), while the mSVF-fibrin hydrogel combination stayed constant at 109.02% (±8.92%). We compared both values obtained on days 3 and 7 in an unpaired t-test with Welch's correction and found no statistically significant difference (p ≥ 0.05).
Histology
In addition, we performed histological analysis with H&E staining (Figure 2). A read-out involving the staining was used as a proof-of-principle, showing the encapsulation of cells in the hydrogel. This served as an overview to demonstrate the overall size of the fibrin scaffold and cellular distribution. There was no visible reduction in the number of cell nuclei at day 3 or day 7, when compared to day 1. The fibrin hydrogel showed little degradation, with visible cell clusters spread evenly throughout the entire construct.
Figure 1: Resazurin assay. The mean percentage value of the Resazurin assay fluorescence intensity in relation to day 0 comparing the mSVF-fibrin hydrogel, the positive control (mSVF), and the negative control (NC, fibrin hydrogel only) over the first 7 days. Bars indicate the mean ± SEM obtained from 4 samples (n = 4). Please click here to view a larger version of this figure.
Figure 2: H&E staining. Representative Image of H&E staining at (A) day 3 and (B) day 7. The scale bar indicates 100 μm. Please click here to view a larger version of this figure.
The mechanical isolation of SVF provides an elegant alternative to the traditional enzymatic approach and offers broad access for clinical application29. In fact, mSVF, as proposed in the present manuscript, is already in clinical use for soft tissue treatment of scars or as an adjunct for cosmetic procedures30. The protocol presented here provides a simple method for efficient topical delivery of viable mSVF cells. While the positive control with only mSVF cells showed a trend toward declining absorption values after 7 days, the mSVF-fibrin hydrogel combination measured steady values. Although not statistically significant, the observed trend remains an important finding. The histology data also showed the number of stained cells to remain constant over the 7-day culture period, demonstrating the cell compatibility of the presented approach. The degradation of the fibrin hydrogels was minimal, and the total size on day 3 and day 7 remained unchanged compared to day 1. In vitro, anti-fibrinolytic additives to the cell culture media, such as tranexamic acid, can be adjusted, allowing the control of degradation times. As shown earlier, emulsification does not completely disrupt all adipocytes, as the histological slides revealed fragments of whole adipocytes embedded in an intact extracellular matrix. This observation was also seen in rat adipose tissue19. Overall, the findings presented here confirm using the fibrin hydrogel as an effective carrier of viable mSVF, for example, for mSVF application in wounds. Combining mSVF with a fibrin hydrogel provides a suitable microenvironment for cellular viability, thereby potentially enhancing the therapeutic efficacy of SVF cells for tissue repair and regeneration purposes22. Furthermore, the processing procedure of adipose tissue to mSVF and the manufacturing of the fibrin hydrogel is a well-established method that can be easily reproduced through this protocol without any major drawbacks19.
Nevertheless, our approach does not come without limitations. Liposuction bears a small possibility of infection, seroma, and systemic complications11,12,31. Of note, the risk increases with increasing volume of liposuction32. In this protocol, only small volumes are needed during the harvesting procedure, therefore minimizing associated risks. The processing of the fat tissue in the protocol provides a simple method without major limitations. A possible limitation of using lipoaspiration for tissue extraction is the potential adverse influence of epinephrine on ASCs. Although research has indicated a cytotoxic effect, the results have been inconsistent33,34,35. In general, lipoaspiration continues to be a dependable and secure method for tissue harvesting31,36,37. The only critical step within the protocol represents the last step of producing the mSVF-fibrin hydrogel-mixture: after the fibrinogen is added, the mSVF-fibrin hydrogel-mixture needs to be applied within approximately 10-30 s due to rapid polymerization, thus limiting the possibility for post-application molding of the construct. It is, therefore, essential to add fibrinogen as the last step and apply the gel mixture in a timely fashion. By altering the thrombin concentration, it is possible to influence the gelation process, leading to either a faster or slower polymerization and a tight or loose interconnected network of fibers22.
While the regenerative potential of ASCs in mSVF has been widely recognized, the application in the clinical scenario has yet to be fully realized. In most investigations, the isolated stromal cells were used without the protection of additional components, leaving the transplanted cells at risk for rapid elimination with poor cell retention, mostly due to interaction with the immune system38. The combination of mSVF with carrier substances can prevent those issues by acting as a scaffold for cell delivery, providing a suitable environment for their survival and proliferation39. A few SVF carriers have been identified and implemented, such as hyaluronic acid, chitosan-, collagen-, alginate- and fibrin-hydrogels39,40,41,42,43. Fibrin gel, a degradable biopolymer formed from fibrinogen, has been widely applied in surgical procedures for wound closure, hemostasis, and as a sealant44,42,46,47. Various benefits are offered by the use of fibrin gel, such as providing cell binding sites to enhance cell attachment, migration, and proliferation22. Furthermore, its advantages include a controllable degradation rate, outstanding biocompatibility, high adhesive properties, and high cell seeding efficiency23,24,25. The combination of those characteristics makes fibrin gel an effective cell carrier for tissue regeneration22. In our mSVF-fibrin hydrogel combination, we observed high cell viability over 7 days, measured by the resazurin assay, as an indicator of metabolic function and cellular health48.
The purpose of this manuscript was to show a simple protocol for encompassing mechanically isolated SVF in a soft, fibrin-based hydrogel. The in vitro testing was performed to demonstrate the durability over 1 week, overall cell distribution, and viability of this method. The presented protocol for an mSVF-fibrin hydrogel offers an alternative to previous mSVF delivery approaches for wound healing or other tissue engineering approaches.
The authors have nothing to disclose.
Bong-Sung Kim is supported by the German Research Foundation (KI 1973/2-1) and the Novartis Foundation for Medical-Biological Research (#22A046).
12-Wellplate | Sarstedt | 83.3921 | |
4′,6-diamidino-2-phenylindole (DAPI) | Biochemica | A1001.0010 | |
50 mL-Falcon | Falcon | 352070 | |
Absorbent Towels, Two Pack | Halyard | 89701 | |
Alamar blue 25 mL | Invitrogen | DAL1025 | |
Albumin, Bovine (BSA) | VWR | 0332-500G | |
Biotek Cytation 5 | Agilent | Cell Imaging Multimode Microplate Reader | |
CaCl2 | Sigma-Aldrich | C5670-500G | |
Cryostat | Microtome | ||
DMEM with 4,5 g/L glucose,with L-Glutamine, with sodium pyruvate | VWR | 392-0416 | |
DPBS | Gibco | 14190-144 | |
Epinephrin | Sigma-Aldrich | E4250 | |
Fetal Bovine Serum | Biowest | S181H-500 | |
Fibrinogen Human Plasma 100 mg | Sigma-Aldrich | 341576-100MG | |
Formalin | Fisher Scientific | SF100-4 | |
Formalin 4% | Formafix | 1308069 | |
FSC 22-Einbettmedium, blau | Biosystems | 3801481S | |
Hematoxylin & Eosin Solution | Sigma-Aldrich | H3136 / HT110132 | |
Lactated Ringer’s Solution 1000 mL | B Braun | R5410-01 | |
Mercedes Cannula 4mm | MicroAire | PAL-R404LL | |
NaCl 0.9% | Bbraun | 570160 | |
OCT Embedding Matrix 125 mL | CellPath | KMA-0100-00A | |
Paraformaldehyde | Fisher Scientific | 10342243 | |
PBS 1% | Sigma-Aldrich | P4474 | |
PenStrep | Sigma-Aldrich | P4333-100ML | |
Petridish 150mm | Sarstedt | 83.1803 | |
Phalloidin-iFluor 488 Reagent | Abcam | ab176753 | |
Sterile Syringe 20 mL Luer | HENKE-JECT | 5200-000V0 | |
Sterile Syringe 30 mL Luer-Lock | BD | 10521 | |
Thrombin from Human Plasma | Sigma-Aldrich | T6884-100UN | |
Tranexamic acid | Orpha Swiss | 6837093 | |
Tulipfilter 1.2 | Lencion Surgical | ATLLLL | |
Tulipfilter 1.4 | Lencion Surgical | ATLLLL |