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 ...

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

Research

JoVE Journal

Medicine

563 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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering 

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Bioengineering

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 ...

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 ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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, ...

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 ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

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

Summary

Explore More Videos

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

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

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

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

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

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Mechanical Micronization of Lipoaspirates for Regenerative Therapy