Name: the combination of mechanically isolated stromal vascular fraction and fibrin hydrogel: a processing protocol
Uploaded: 2023-11-17T12:00:00.000+00:00
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Description: Read this Scientific Article Protocol about The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol at JoVE.com

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><tbody><tr><td>12-Wellplate</td><td>Sarstedt</td><td>83.3921</td><td></td></tr><tr><td>4&amp;prime;,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&amp;nbsp;</td><td>Agilent</td><td></td><td>Cell Imaging Multimode Microplate Reader&amp;nbsp;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</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 &amp;amp; Eosin Solution</td><td>Sigma-Aldrich</td><td>H3136 / HT110132</td><td></td></tr><tr><td>Lactated Ringer&amp;rsquo;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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Research

JoVE Journal

Medicine

495 Views.  Triemli City Hospital Zurich. Chronische wonden en huiddefecten in een groot gebied vormen een grote klinische last met beperkte therapeutische opties. We streven ernaar om het gebruik van een direct beschikbare autologe celbron, vetweefsel, te onderzoeken als een mogelijke behandelingsmogelijkheid. Stromale vasculaire fractie wordt geïsoleerd uit vetweefsel verkregen door excisie of liposuctie.Mechanisch geïsoleerde stromale vasculaire fractie wordt gebruikt als alternatief voor...

Video: Auteur in de schijnwerpers: onderzoek naar het potentieel van van vet afgeleide stromale vasculaire fractie voor wondgenezing

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Bioengineering

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Developmental Biology

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

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

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