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.

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

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

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 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 ...

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476 Views.  Triemli City Hospital Zurich. 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.

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

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...