The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 t…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Bong-Sung Kim is supported by the German Research Foundation (KI 1973/2-1) and the Novartis Foundation for Medical-Biological Research (#22A046).
Materials
| 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 |
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