The authors acknowledge funding from NIH R00 HL115123 and Brown University School of Engineering. They are also grateful to the Brown Design Workshop and Chris Bull for training and support with the laser cutter.

<ol>
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Rev. 101, 1869-1880 (2001).</li><li>Kloxin, A., Kloxin, C., Bowman, C., Anseth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+cellularly+responsive+hydrogels+and+their+experimental+determination.">Mechanical properties of cellularly responsive hydrogels and their experimental determination.</a> Adv. Mater. Deerfield Beach Fla. 22, 3484-3494 (2010).</li><li>Aubin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+3D+cell+alignment+and+elongation+in+microengineered+hydrogels.">Directed 3D cell alignment and elongation in microengineered hydrogels.</a> Biomaterials. 31, 6941-6951 (2010).</li><li>Jaiswal, M. 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Protoc. 8, 162-175 (2013).</li><li>Boxshall, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+surface+treatments+to+modify+protein+adsorption+and+cell+attachment+properties+within+a+poly(dimethylsiloxane)+micro-bioreactor.">Simple surface treatments to modify protein adsorption and cell attachment properties within a poly(dimethylsiloxane) micro-bioreactor.</a> Surf. Interface Anal. 38, 198-201 (2006).</li><li>Pins, G. D., Christiansen, D. L., Patel, R., Silver, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+collagen+fibers.+Influence+of+fibrillar+alignment+and+decorin+on+mechanical+properties.">Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.</a> Biophys. J. 73, 2164-2172 (1997).</li><li>Pipan, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+antifibrinolytic+agents+on+the+life+span+of+fibrin+sealant.">Effects of antifibrinolytic agents on the life span of fibrin sealant.</a> J. Surg. Res. 53, 402-407 (1992).</li><li>Roberts, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Cells+in+Dense+Collagen+Promote+Cardiomyocyte+and+Microvascular+Patterning+in+Engineered+Human+Heart+Tissue.">Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.</a> Tissue Eng. Part A. 22, 633-644 (2016).</li><li>Ye, K. Y., Sullivan, K. E., Black, L. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-Etched+Designs+for+Molding+Hydrogel-Based+Engineered+Tissues.">Laser-Etched Designs for Molding Hydrogel-Based Engineered Tissues.</a> Tissue Eng. Part C Methods. 23, 311-321 (2017).</li><li>Zhang, H., Chiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fouling+Coatings+of+Poly(dimethylsiloxane)+Devices+for+Biological+and+Biomedical+Applications.">Anti-fouling Coatings of Poly(dimethylsiloxane) Devices for Biological and Biomedical Applications.</a> J. Med. Biol. Eng. 35, 143-155 (2015).</li></ol>

The authors have nothing to disclose.

Customized PDMS mold geometries that are compatible with tissue culture have great utility in tuning important engineered tissue properties, such as cell alignment, diffusion rate, and effective stiffness. Additionally, these molds are very useful for preparing tissues for analysis applications in which geometry is important, such as mechanical testing16,17. Preparing these devices from laser cut negative master molds offers a rapid, facile, and low-cost method o...

Over the past two decades, soft lithography has been used extensively as a fabrication technique to support scientific research, particularly in the fields of microfluidics, materials research, and tissue engineering1,2,3. Replica molding, in which an object with a desired shape is created from a negative master mold, offers a convenient and low-cost method of producing positive PDMS replicates that can be used for casting shaped hydrogels. However, the required negative master molds are typically produced using microfabrication techniques that are expensive, time-consuming, limited in size, and require clean room space and sophisticated equipment. While 3D printing offers a potential alternative, its utility is somewhat limited due to the resolution limits of lower-cost printers and the chemical interactions between common 3D printer polymers and PDMS that can inhibit curing.
Laser cutter systems capable of both cutting and etching materials such as plastic, wood, glass, and metal have recently become drastically less expensive and therefore more accessible for fabricating research tools. Commercial grade laser cutters are capable of fabricating objects on the centimeter scale with minimum features smaller than 25 &#181;m, and further require minimal training, expertise, and time to use. While laser ablation of PDMS has been previously used in the fabrication of microfluidics devices, to our knowledge no manuscript has described a process by which millimeter and centimeter scale molds can be fabricated from laser cut negative master molds4.
We have used this technique primarily to manipulate the shape of engineered tissues in order to improve nutrient diffusion, cellular alignment, and mechanical properties5,6,7. However, versatility of this technique allows for the utilization in any field where molded hydrogels are of interest, such as drug delivery and material science research8. With access to a laser cutter, PDMS mold replicates can be made for nearly any geometry without overhangs (that would inhibit removal without a multi-part mold, which is beyond the scope of this manuscript) and that fits within the dimensions of the laser bed.

<table border="0" cellpadding="0" cellspacing="0" width="738">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Item</td>
			<td style="width:167px;"></td>
			<td style="width:169px;"></td>
			<td style="width:141px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630-5G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine thrombin</td>
			<td>Sigma</td>
			<td>T6634-250UN</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine aprotinin</td>
			<td>Sigma</td>
			<td>10820-25MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I, 4 mg/mL</td>
			<td>Advanced Biomatrix</td>
			<td>5153-100MG</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodim chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicone elastomer</td>
			<td>Corning</td>
			<td>4019862</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tape</td>
			<td>Fisher</td>
			<td>15-901-5R</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylic, 1/4&#34; thick</td>
			<td>McMaster-Carr</td>
			<td>8560K356</td>
			<td>PDMS Molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffer, 1 M</td>
			<td>Sigma</td>
			<td>H3537-100ML</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 medium, powder</td>
			<td>Fisher</td>
			<td>31800-089</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>Fisher</td>
			<td>AC423520250</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Fisher</td>
			<td>M33 500</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate dibasic heptahydrate</td>
			<td>Sigma</td>
			<td>S9390-500G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G5767-25G</td>
			<td>Constructs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Frozen block molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin</td>
			<td>Fisher</td>
			<td>3530 1</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y</td>
			<td>Fisher</td>
			<td>AC152880250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast green FCF</td>
			<td>Fisher</td>
			<td>AC410530250</td>
			<td>Histology</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Illustrator</td>
			<td>Adobe Systems</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inkscape</td>
			<td>(Open Source)</td>
			<td></td>
			<td>Vector Graphics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UCP (Universal Control Panel)</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter Interface</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PLS6.75 Laser Cutter</td>
			<td>Universal Laser Systems</td>
			<td></td>
			<td>Laser Cutter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromechanical Analyzer</td>
			<td>Aurora Scientific</td>
			<td>1530A with 5 mN load cell</td>
			<td>Mechanical Analysis</td>
		</tr>
	</tbody>
</table>

custom engineered tissue culture molds from laser-etched masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

The optics of the laser cutter will cause etched areas to have very slightly decreased dimensions as etching depth increases, and results in mold walls with a very subtle bevel, due to tapering of the laser beam. This will help facilitate the removal of the cast PDMS molds, but should be carefully considered if very deeply etched negative master molds (&#62;6 mm) are required (Figure 1).
Over time i...

Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

Custom Engineered Tissue Culture Molds from Laser-etched Masters

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

custom-engineered-tissue-culture-molds-from-laser-etched-masters

Research

JoVE Journal

Bioengineering

6.4K Views.  Brown University. Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

Video: Custom Engineered Tissue Culture Molds from Laser-etched Masters

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...