<meta charset="utf8"></head><body>再生医療のためのフィブリンハイドロゲル中の機械的間質血管分画

<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

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.

<meta charset="utf8"></head><body>著者スポットライト:創傷治癒のための脂肪由来間質血管画分の可能性の探求

機械的に単離されたStromal Vascular画分およびFibrinのヒドロゲルの組合せ:処理のプロトコール

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.

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

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

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Research

JoVE Journal

Medicine

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

366 ビュー.  Triemli City Hospital Zurich. 慢性創傷や広範囲の皮膚欠損は、治療の選択肢が限られているため、大きな臨床的負担となっています。私たちは、治療の機会として、容易に利用できる自家細胞源である脂肪組織の使用を調査することを目指しています。間質血管画分は、切除または脂肪吸引によって得られた脂肪組織から分離されます。機械的に分離された間質血管画...

ビデオ: 著者スポットライト:創傷治癒のための脂肪由来間質血管画分の可能性の探求

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.

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.

医学

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.