Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
JoVE Journal Medicine

Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

Published: November 17, 2023 doi: 10.3791/65860
*1, *2, 2, 2, 2, 3, 2
1Department of Surgery, Triemli City Hospital Zurich, 2Department of Plastic Surgery and Hand Surgery, University Hospital Zurich, University of Zurich, 3Institute for Human Tissue Restoration, Department of Plastic and Reconstructive Surgery, Yonsei University College of Medicine
* These authors contributed equally

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.

Subscription Required. Please recommend JoVE to your librarian.

Protocol

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

  1. Harvest the adipose tissue by performing a standard liposuction in a conventional fat-harvesting technique described in previous publications26,27. Ensure to use a tumescent solution that consists of regular ringer's lactate and epinephrine in a ratio of 1: 200,000.
  2. Perform the liposuction with a 4 mm aspiration cannula, under negative pressure and vibration, into a sterile bag. Transfer the harvested fat to the laboratory immediately.

2. mSVF-Isolation

  1. Perform the following steps (sections 2 and 3) in a cell culture hood to provide an aseptic work area. Wear a regular lab coat and gloves to ensure biological safety level 2.
  2. Prepare the culture medium: Supplement 500 mL of high-glucose Dulbecco's Modified Eagles's Medium (DMEM) with 50 mL of fetal bovine serum (FBS), 5 mL of penicillin-streptomycin.
  3. Transfer the lipoaspirate into a 50 mL centrifuge tube.
  4. Transfer the lipoaspirate into a sterile 20 mL Luer-lock syringe and attach a 1.4 mm connector. Ensure that no air is inside the syringe.
  5. Attach a second 20 mL Luer-lock syringe to the contralateral side of the 1.4 mm connector.
  6. Push the adipose tissue from one syringe to the other for a total of 30 times.
  7. Transfer the emulsified fat into a fresh 50 mL centrifuge tube.
  8. Centrifugate the emulsified fat at 500 x g for 10 min.
  9. After centrifugation, discard the oily top layer. Then, collect the central purified mSV-layer. Transfer it into a fresh 50 mL centrifuge tube and discard the aqueous phase.
  10. Fill the centrifuge tube with culture medium (from step 2.2) up to the 40 mL mark.
  11. Place the centrifuge tube into the centrifuge and once again centrifuge at 500 x g for 5 min.
  12. Collect the resulting mSVF-layer and transfer it into a new 50 mL centrifuge tube.

3. Manufacturing of mSVF-fibrin hydrogel

  1. Combine 100 µL of mSVF with 10 µL of thrombin (100 U/mL), 10 µL of CaCl2 (80 mM), and 70 µL of tranexamic acid (100 mg/mL) in a sterile 1.5 mL tube.
  2. Use a fresh pipette tip to add 10 µL of fibrinogen (100 mg/mL) as the last component, shortly before application. Beware that hydrogel-polymerization is observed within approximately 10-30 s.
  3. For clinical application, perform this last step shortly before topical administration, preferably at the bedside. For analytical purposes, transfer into a 12-well plate by pipetting.

4. Viability assay and histology

  1. Pipette 200 µL of the mSVF-hydrogel mixture from step 3.3 into one well of a 12-well plate.
  2. Pipette 100 µL of the mSVF-collection (step 2.12.) into one well as a positive control.
  3. Pipette 200 µL of fibrin hydrogel only into one well as a negative control.
  4. Place the 12-well plate into a regular incubator at 37 °C and 5% CO2 for 30 min.
  5. After this time, add 1 mL of resazurin (alamar blue, 10% concentration, diluted in culture medium) to each well.
  6. After the addition, incubate the 12 well-plate at 37 °C and 5% CO2 for 24 h.
  7. After 24 h, measure the first fluorescence intensity using a cell imaging multimode microplate reader using an excitation wavelength of 555 nm and an emission wavelength of 596 nm.
  8. Perform consecutive fluorescence intensity measurements on days 3 and 7.
  9. If histologic assessment is necessary, stop the experiment on days 1, 3, and 7 and fix the fibrin hydrogel in 4% paraformaldehyde at 4 °C for 24 h. After fixation, add 1% phosphate-buffered saline (PBS) and store it at 4 °C.
  10. Embed the hydrogels in an optimal cutting temperature (OCT) compound and section frozen blocks at 10 µm thickness with a cryostat. Stain the sections with hematoxylin and eosin (H&E) solution according to standard protocols28.

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

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
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
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.

Subscription Required. Please recommend JoVE to your librarian.

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 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.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors have nothing to disclose.

Acknowledgments

Bong-Sung Kim is supported by the German Research Foundation (KI 1973/2-1) and the Novartis Foundation for Medical-Biological Research (#22A046).

Materials

Name Company Catalog Number Comments
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

DOWNLOAD MATERIALS LIST

References

  1. Daar, A. S., Greenwood, H. L. A proposed definition of regenerative medicine. J Tissue Eng Regen Med. 1 (3), 179-184 (2007).
  2. Machens, H. G., Mailänder, P. Regenerative medicine and plastic surgery. Chirurg. 76 (5), 474-480 (2005).
  3. Zuk, P. A., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7 (2), 211-228 (2001).
  4. Hassan, W. U., Greiser, U., Wang, W. Role of adipose-derived stem cells in wound healing. Wound Repair Regen. 22 (3), 313-325 (2014).
  5. Gir, P., Oni, G., Brown, S. A., Mojallal, A., Rohrich, R. J. Human adipose stem cells: current clinical applications. Plast Reconstr Surg. 129 (6), 1277-1290 (2012).
  6. Dominici, M., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8 (4), 315-317 (2006).
  7. Raposio, E., Ciliberti, R. Clinical use of adipose-derived stem cells: European legislative issues. Ann Med Surg (Lond). 24, 61-64 (2017).
  8. Condé-Green, A., et al. Shift toward mechanical isolation of adipose-derived stromal vascular fraction: Review of upcoming techniques. Plast Reconstr Surg Glob Open. 4 (9), e1017 (2016).
  9. Dykstra, J. A., et al. Concise review: Fat and furious: Harnessing the full potential of adipose-derived stromal vascular fraction. Stem Cells Transl Med. 6 (4), 1096-1108 (2017).
  10. Aronowitz, J. A., Lockhart, R. A., Hakakian, C. S. Mechanical versus enzymatic isolation of stromal vascular fraction cells from adipose tissue. Springerplus. 4, 713 (2015).
  11. Tonnard, P., et al. Nanofat grafting: basic research and clinical applications. Plast Reconstr Surg. 132 (4), 1017-1026 (2013).
  12. Tiryaki, K. T., Cohen, S., Kocak, P., Canikyan Turkay, S., Hewett, S. In-vitro comparative examination of the effect of stromal vascular fraction isolated by mechanical and enzymatic methods on wound healing. Aesthet Surg J. 40 (11), 1232-1240 (2020).
  13. Sun, M., et al. Adipose extracellular matrix/stromal vascular fraction gel secretes angiogenic factors and enhances skin wound healing in a murine model. Biomed Res Int. 2017, 3105780 (2017).
  14. Chen, L., et al. Autologous nanofat transplantation accelerates foot wound healing in diabetic rats. Regen Med. 14 (3), 231-241 (2019).
  15. Lin, Y. N., et al. Fat grafting for resurfacing an exposed implant in lower extremity: A case report. Medicine (Baltimore). 96 (48), e8901 (2017).
  16. Deng, C., Wang, L., Feng, J., Lu, F. Treatment of human chronic wounds with autologous extracellular matrix/stromal vascular fraction gel: A STROBE-compliant study. Medicine (Baltimore). 97 (32), e11667 (2018).
  17. Chung, M. T., et al. Micro-computed tomography evaluation of human fat grafts in nude mice. Tissue Eng Part C Methods. 19 (3), 227-232 (2013).
  18. Gonzalez, A. M., Lobocki, C., Kelly, C. P., Jackson, I. T. An alternative method for harvest and processing fat grafts: an in vitro study of cell viability and survival. Plast Reconstr Surg. 120 (1), 285-294 (2007).
  19. Kim, B. S., et al. In vivo evaluation of mechanically processed stromal vascular fraction in a chamber vascularized by an arteriovenous shunt. Pharmaceutics. 14 (2), 417 (2022).
  20. Dryden, G. W., Boland, E., Yajnik, V., Williams, S. Comparison of stromal vascular fraction with or without a novel bioscaffold to fibrin glue in a porcine model of mechanically induced anorectal fistula. Inflamm Bowel Dis. 23 (11), 1962-1971 (2017).
  21. Mahoney, C. M., Imbarlina, C., Yates, C. C., Marra, K. G. Current therapeutic strategies for adipose tissue defects/repair using engineered biomaterials and biomolecule formulations. Front Pharmacol. 9, 507 (2018).
  22. Li, Y., Meng, H., Liu, Y., Lee, B. P. Fibrin gel as an injectable biodegradable scaffold and cell carrier for tissue engineering. ScientificWorldJournal. 2015, 685690 (2015).
  23. Malafaya, P. B., Silva, G. A., Reis, R. L. Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Adv Drug Deliv Rev. 59 (4-5), 207-233 (2007).
  24. Jackson, M. R. Fibrin sealants in surgical practice: An overview. Am J Surg. 182 (2 Suppl), 1s-7s (2001).
  25. Swartz, D. D., Russell, J. A., Andreadis, S. T. Engineering of fibrin-based functional and implantable small-diameter blood vessels. Am J Physiol Heart Circ Physiol. 288 (3), H1451-H1460 (2005).
  26. Alharbi, Z., et al. Conventional vs. micro-fat harvesting: how fat harvesting technique affects tissue-engineering approaches using adipose tissue-derived stem/stromal cells. J Plast Reconstr Aesthet Surg. 66 (9), 1271-1278 (2013).
  27. Coleman, S. R. Structural fat grafts: the ideal filler. Clin Plast Surg. 28 (1), 111-119 (2001).
  28. Feldman, A. T., Wolfe, D. Tissue processing and hematoxylin and eosin staining. Methods Mol Biol. 1180, 31-43 (2014).
  29. Raposio, E., Bertozzi, N. How to isolate a ready-to-use adipose-derived stem cells pellet for clinical application. Eur Rev Med Pharmacol Sci. 21 (18), 4252-4260 (2017).
  30. Pallua, N., Kim, B. S. Microfat and lipoconcentrate for the treatment of facial scars. Clin Plast Surg. 47 (1), 139-145 (2020).
  31. Pallua, N., Grasys, J., Kim, B. S. Enhancement of progenitor cells by two-step centrifugation of emulsified lipoaspirates. Plast Reconstr Surg. 142 (1), 99-109 (2018).
  32. Lehnhardt, M., et al. Major and lethal complications of liposuction: a review of 72 cases in Germany between 1998 and 2002. Plast Reconstr Surg. 121 (6), 396e-403e (2008).
  33. Shoshani, O., et al. The effect of lidocaine and adrenaline on the viability of injected adipose tissue--an experimental study in nude mice. J Drugs Dermatol. 4 (3), 311-316 (2005).
  34. Girard, A. C., et al. New insights into lidocaine and adrenaline effects on human adipose stem cells. Aesthetic Plast Surg. 37 (1), 144-152 (2013).
  35. Grambow, F., et al. The impact of lidocaine on adipose-derived stem cells in human adipose tissue harvested by liposuction and used for lipotransfer. Int J Mol Sci. 21 (8), 2869 (2020).
  36. Xiao, S., et al. Mechanical micronization of lipoaspirates combined with fractional CO2 laser for the treatment of hypertrophic scars. Plast Reconstr Surg. 151 (3), 549-559 (2023).
  37. Zhang, J., et al. Adipose tissue-derived pericytes for cartilage tissue engineering. Curr Stem Cell Res Ther. 12 (6), 513-521 (2017).
  38. Yao, Y., et al. Adipose extracellular matrix/stromal vascular fraction gel: A novel adipose tissue-derived injectable for stem cell therapy. Plast Reconstr Surg. 139 (4), 867-879 (2017).
  39. Williams, S. K., Touroo, J. S., Church, K. H., Hoying, J. B. Encapsulation of adipose stromal vascular fraction cells in alginate hydrogel spheroids using a direct-write three-dimensional printing system. Biores Open Access. 2 (6), 448-454 (2013).
  40. Denost, Q., et al. Colorectal wall regeneration resulting from the association of chitosan hydrogel and stromal vascular fraction from adipose tissue. J Biomed Mater Res A. 106 (2), 460-467 (2018).
  41. Nilforoushzadeh, M. A., et al. Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin-collagen hydrogel: A clinical study for diabetic wound healing. J Tissue Eng Regen Med. 14 (3), 424-440 (2020).
  42. Lin, S. D., et al. Injected implant of uncultured stromal vascular fraction loaded onto a collagen gel: In vivo study of adipogenesis and long-term outcomes. Ann Plast Surg. 76 (Suppl 1), S108-S116 (2016).
  43. Lv, X., et al. Comparative efficacy of autologous stromal vascular fraction and autologous adipose-derived mesenchymal stem cells combined with hyaluronic acid for the treatment of sheep osteoarthritis. Cell Transplant. 27 (7), 1111-1125 (2018).
  44. de Boer, M. T., Boonstra, E. A., Lisman, T., Porte, R. J. Role of fibrin sealants in liver surgery. Dig Surg. 29 (1), 54-61 (2012).
  45. Fuller, C. Reduction of intraoperative air leaks with Progel in pulmonary resection: a comprehensive review. J Cardiothorac Surg. 8, 90 (2013).
  46. Ratnalingam, V., Eu, A. L., Ng, G. L., Taharin, R., John, E. Fibrin adhesive is better than sutures in pterygium surgery. Cornea. 29 (5), 485-489 (2010).
  47. Sierra, D. H. Fibrin sealant adhesive systems: a review of their chemistry, material properties and clinical applications. J Biomater Appl. 7 (4), 309-352 (1993).
  48. Rampersad, S. N. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel). 12 (9), 12347-12360 (2012).

Tags

Medicine
This article has been published
Video Coming Soon
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Pfister, P., Reid, G., Breckwoldt,… More

Pfister, P., Reid, G., Breckwoldt, T., Vasella, M., Seitz, A. K., Song, S. Y., Kim, B. S. The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol. J. Vis. Exp. (201), e65860, doi:10.3791/65860 (2023).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter