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

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&#39;s human research ethics committee.
1. Harvest of adipose tissue
<ol>
	<li>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&#39;s lactate and epinephrine in a ratio of 1: 200,000.</li>
	<li>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.</li>
</ol>
2. mSVF-Isolation
<ol>
	<li>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.</li>
	<li>Prepare the culture medium: Supplement 500 mL of high-glucose Dulbecco&#39;s Modified Eagles&#39;s Medium (DMEM) with 50 mL of fetal bovine serum (FBS), 5 mL of penicillin-streptomycin.</li>
	<li>Transfer the lipoaspirate into a 50 mL centrifuge tube.</li>
	<li>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.</li>
	<li>Attach a second 20 mL Luer-lock syringe to the contralateral side of the 1.4 mm connector.</li>
	<li>Push the adipose tissue from one syringe to the other for a total of 30 times.</li>
	<li>Transfer the emulsified fat into a fresh 50 mL centrifuge tube.</li>
	<li>Centrifugate the emulsified fat at 500 x g for 10 min.</li>
	<li>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.</li>
	<li>Fill the centrifuge tube with culture medium (from step 2.2) up to the 40 mL mark.</li>
	<li>Place the centrifuge tube into the centrifuge and once again centrifuge at 500 x g for 5 min.</li>
	<li>Collect the resulting mSVF-layer and transfer it into a new 50 mL centrifuge tube.</li>
</ol>
3. Manufacturing of mSVF-fibrin hydrogel
<ol>
	<li>Combine 100 &#181;L of mSVF with 10 &#181;L of thrombin (100 U/mL), 10 &#181;L of CaCl2 (80 mM), and 70 &#181;L of tranexamic acid (100 mg/mL) in a sterile 1.5 mL tube.</li>
	<li>Use a fresh pipette tip to add 10 &#181;L of fibrinogen (100 mg/mL) as the last component, shortly before application. Beware that hydrogel-polymerization is observed within approximately 10-30 s.</li>
	<li>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.</li>
</ol>
4. Viability assay and histology
<ol>
	<li>Pipette 200 &#181;L of the mSVF-hydrogel mixture from step 3.3 into one well of a 12-well plate.</li>
	<li>Pipette 100 &#181;L of the mSVF-collection (step 2.12.) into one well as a positive control.</li>
	<li>Pipette 200 &#181;L of fibrin hydrogel only into one well as a negative control.</li>
	<li>Place the 12-well plate into a regular incubator at 37 &#176;C and 5% CO2 for 30 min.</li>
	<li>After this time, add 1 mL of resazurin (alamar blue, 10% concentration, diluted in culture medium) to each well.</li>
	<li>After the addition, incubate the 12 well-plate at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>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.</li>
	<li>Perform consecutive fluorescence intensity measurements on days 3 and 7.</li>
	<li>If histologic assessment is necessary, stop the experiment on days 1, 3, and 7 and fix the fibrin hydrogel in 4% paraformaldehyde at 4 &#176;C for 24 h. After fixation, add 1% phosphate-buffered saline (PBS) and store it at 4 &#176;C.</li>
	<li>Embed the hydrogels in an optimal cutting temperature (OCT) compound and section frozen blocks at 10 &#181;m thickness with a cryostat. Stain the sections with hematoxylin and eosin (H&#38;E) solution according to standard protocols28.</li>
</ol>

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The authors have nothing to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-Wellplate</td>
			<td>Sarstedt</td>
			<td>83.3921</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Biochemica</td>
			<td>A1001.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL-Falcon</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Towels, Two Pack</td>
			<td>Halyard</td>
			<td>89701</td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar blue 25 mL</td>
			<td>Invitrogen</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine (BSA)</td>
			<td>VWR</td>
			<td>0332-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotek Cytation 5&#160;</td>
			<td>Agilent</td>
			<td></td>
			<td>Cell Imaging Multimode Microplate Reader&#160;</td>
		</tr>
		<tr>
			<td>CaCl2&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C5670-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microtome</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with 4,5 g/L glucose,with L-Glutamine, with sodium pyruvate</td>
			<td>VWR</td>
			<td>392-0416</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Epinephrin</td>
			<td>Sigma-Aldrich</td>
			<td>E4250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>S181H-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen Human Plasma 100 mg</td>
			<td>Sigma-Aldrich</td>
			<td>341576-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Fisher Scientific</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin 4%</td>
			<td>Formafix</td>
			<td>1308069</td>
			<td></td>
		</tr>
		<tr>
			<td>FSC 22-Einbettmedium, blau</td>
			<td>Biosystems</td>
			<td>3801481S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; Eosin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>H3136 / HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#8217;s Solution 1000 mL</td>
			<td>B Braun</td>
			<td>R5410-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercedes Cannula 4mm</td>
			<td>MicroAire</td>
			<td>PAL-R404LL</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Bbraun</td>
			<td>570160</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Embedding Matrix 125 mL</td>
			<td>CellPath</td>
			<td>KMA-0100-00A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>10342243</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1%</td>
			<td>Sigma-Aldrich</td>
			<td>P4474</td>
			<td></td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish 150mm</td>
			<td>Sarstedt</td>
			<td>83.1803</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 488 Reagent</td>
			<td>Abcam</td>
			<td>ab176753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe 20 mL Luer</td>
			<td>HENKE-JECT</td>
			<td>5200-000V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe 30 mL Luer-Lock</td>
			<td>BD</td>
			<td>10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from Human Plasma</td>
			<td>Sigma-Aldrich</td>
			<td>T6884-100UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Tranexamic acid</td>
			<td>Orpha Swiss</td>
			<td>6837093</td>
			<td></td>
		</tr>
		<tr>
			<td>Tulipfilter 1.2</td>
			<td>Lencion Surgical</td>
			<td>ATLLLL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tulipfilter 1.4</td>
			<td>Lencion Surgical</td>
			<td>ATLLLL</td>
			<td></td>
		</tr>
	</tbody>
</table>

the combination of mechanically isolated stromal vascular fraction and fibrin hydrogel: a processing protocol

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.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

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

Chronic wounds and large area skin defects pose a major clinical burden with limited therapeutic options. We aim to investigate the use of a readily available autologous cell source, fat tissue, as a possible treatment opportunity. Stromal vascular fraction is isolated from fat tissue obtained either by excision or liposuction.Mechanically isolated stromal vascular fraction is used as an alternative to enzymatically processed SVF in order to bypass the problems faced by extra corporeal laboratory processing. So typical problems include long processing time, risk of contamination, and incurred costs. 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. In future, we will address how to translate our acquired knowledge into a clinical setting, specifically the effect of each cell type represented in SVF in in vitro pathological condition.

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% (&#177; 5.33%), while the mSVF-fibrin hydrogel combination remained at 9...

Read this Scientific Article Protocol about The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol at JoVE.com

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 ...

the-combination-mechanically-isolated-stromal-vascular-fraction

Research

JoVE Journal

Medicine

476 Views.  Triemli City Hospital Zurich. Le ferite croniche e i difetti cutanei di vaste aree rappresentano un importante onere clinico con opzioni terapeutiche limitate. Il nostro obiettivo è quello di studiare l'uso di una fonte di cellule autologhe prontamente disponibile, il tessuto adiposo, come possibile opportunità di trattamento. La frazione vascolare stromale è isolata dal tessuto adiposo ottenuto mediante escissione o liposuzione.La frazione vascolare stromale isolata meccanicame...

Video: Autore in primo piano: Esplorare il potenziale della frazione vascolare stromale derivata dal grasso per la guarigione delle ferite

Mechanical Stromal Vascular Fraction (mSVF) Isolation from Lipoaspirate and its Fibrin Hydrogel Production for Clinical Application

To begin, in a cell culture hood, add the harvested lipoaspirate into a 50 milliliter centrifuge tube. Transfer the lipoaspirate into a sterile 20 milliliter Luer lock syringe. Then attach a 1.4 millimeter connector to the syringe.Now attach a second 20 milliliter Luer lock syringe to the contralateral side of the connector. Push the adipose tissue from one syringe to the other about 30 times. Then transfer the emulsified fat into a fresh 50 milliliter centrifuge tube.Centrifugate the fat at 500 G for 10 minutes. Then discard the oily top layer. Collect the central purified layer containing the separated stromal vascular layer and transfer it into a fresh 50 milliliter centrifuge tube.Now fill the centrifuge tube with supplemented DMEM up to the 40 milliliter mark. Centrifuge the tube again at 500 G for five minutes. Collect the resulting mSVF layer and transfer it into a new 50 milliliter centrifuge tube.Into a sterile 1.5 milliliter tube, pipette 100 microliters of the isolated stromal vascular fraction. To this, add 10 microliters of thrombin, then pipette 10 microliters of calcium chloride. Finally, add 70 microliters of tranexamic acid to the mix.With a fresh pipette tip, add 10 microliters of fibrinogen to the tube shortly before application. After the polymerization of the mSVF hydrogel mixture, pipette 200 microliters of the hydrogel into a 12-well plate for analytical purposes. Now add 100 microliters of the fibrin hydrogel into one well as a negative control.Incubate the 12-well plate at 37 degrees Celsius under 5%carbon dioxide for 30 minutes. Then add one milliliter of pre-warmed culture medium to each well. Place the 12-well plate back into the incubator again for four hours.Add one milliliter resazurin to each well before incubating again for 24 hours. Pipette the resazurin samples into a 96-well plate. The next day, use a cell imaging multi-mode microplate reader to measure the first fluorescence intensity.For histological analysis on days one, three, and seven, incubate the fibrin hydrogel in 0.5 milliliters of 4%paraformaldehyde. Now add 1%PBS to the plate and store it at four degrees Celsius. The resazurin assay was performed to quantify the in vitro cell viabilities of the vascular fraction and the hydrogel.The vascular fraction showed reduced viability on day three, whereas the mSVF-fibrin hydrogel combination remained close to baseline. By day seven, the viability of the mSVF dropped, while the mSVF-fibrin hydrogel combination increased. Histological analysis showed no reduction in the number of cell nuclei on days three and seven.The fibrin hydrogel displayed minimal degradation with evenly distributed visible cell clusters.