Name: custom engineered tissue culture molds from laser-etched masters
Uploaded: 2018-05-21T13:00:00
Description: Watch this Scientific Journal Video about Custom Engineered Tissue Culture Molds from Laser-etched Masters at JoVE.com

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.

1. Create the Vector Format Master Mold Designs
<ol>
	<li>Assemble the desired mold geometry in vector format using a vector graphics program (see Materials, Equipment, and Software Table). Select File| New and create a canvas of appropriate dimensions with RGB color format. Create the desired geometry using the shape tools in the left-hand panel: enter the desired dimensions at the top of the window (click the transform button at the top if not initially visible) to precisely define s...

<ol>
	<li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscale+patterning.">Soft lithography for micro- and nanoscale patterning.</a> Nat. Protoc. 5, 491 (2010).</li><li>Rogers, J. A., Nuzzo, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+soft+lithography.">Recent progress in soft lithography.</a> Mater. Today. 8, 50-56 (2005).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography+in+Biology+and+Biochemistry.">Soft Lithography in Biology and Biochemistry.</a> Annu. Rev. Biomed. Eng. 3, 335-373 (2001).</li><li>Isiksacan, Z., Guler, M. T., Aydogdu, B., Bilican, I., Elbuken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+fabrication+of+microfluidic+PDMS+devices+from+reusable+PDMS+molds+using+laser+ablation.">Rapid fabrication of microfluidic PDMS devices from reusable PDMS molds using laser ablation.</a> J. Micromechanics Microengineering. 26, 035008 (2016).</li><li>Lee, K. Y., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+Tissue+Engineering.">Hydrogels for Tissue Engineering.</a> Chem. 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. 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=Vacancy-Driven+Gelation+Using+Defect-Rich+Nanoassemblies+of+2D+Transition+Metal+Dichalcogenides+and+Polymeric+Binder+for+Biomedical+Applications.">Vacancy-Driven Gelation Using Defect-Rich Nanoassemblies of 2D Transition Metal Dichalcogenides and Polymeric Binder for Biomedical Applications.</a> Adv. Mater. 29, (2017).</li><li>Lian, X., 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+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+by+modulating+Wnt/&#946;-catenin+signaling+under+fully+defined+conditions.">Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/&#946;-catenin signaling under fully defined conditions.</a> Nat. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+Cardiomyocytes+in+a+Fibrin+Hydrogel+for+Cardiac+Tissue+Engineering.">Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering.</a> JoVE. , (2011).</li><li>Zimmermann, W. 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=Tissue+Engineering+of+a+Differentiated+Cardiac+Muscle+Construct.">Tissue Engineering of a Differentiated Cardiac Muscle Construct.</a> Circ. Res. 90, 223-230 (2002).</li><li>McCain, M. L., Agarwal, A., Nesmith, H. W., Nesmith, A. P., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolded+Gelatin+Hydrogels+for+Extended+Culture+of+Engineered+Cardiac+Tissues.">Micromolded Gelatin Hydrogels for Extended Culture of Engineered Cardiac Tissues.</a> Biomaterials. 35, 5462-5471 (2014).</li><li>Hu, J. J., Chen, G. W., Liu, Y. C., Hsu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Specimen+Geometry+on+the+Estimation+of+the+Planar+Biaxial+Mechanical+Properties+of+Cruciform+Specimens.">Influence of Specimen Geometry on the Estimation of the Planar Biaxial Mechanical Properties of Cruciform Specimens.</a> Exp. Mech. 54, 615-631 (2014).</li><li>Munarin, F., Kaiser, N. J., Kim, T. Y., Choi, B. R., Coulombe, K. L. 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>

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 ...

This method can facilitate research in the tissue-engineering field related to tissue development, cell alignment, and mechanical analysis of polymers and engineered tissues. The main advantage of this technique is that it is low-cost, easy to use, and fast. We developed this technique in my lab because geometric control of engineered tissues on the millimeter-to-centimeter scale was not being done in the field, and we saw a critical need to think translationally about scaling up the size of engineered tissues.This technique allows us to leverage the benefits of geometric queues on cellular physiology and engineered tissues through fabrication techniques using posts and a variety of shapes. This method can provide insight into the production of engineered tissues with controlled size, shape, and aligned. It can also be applied to various disease models, personalized regenerative medicine, and for the translation of engineered tissues to preclinical studies.Begin by opening a vector graphics program on a computer. To prepare the desired mold geometry and vector format, open a new canvas of appropriate dimensions with the RGB color format. Then, select the shape tool from the left-hand panel and create the desired geometry by entering the dimensions at the top of the window to precisely define shape sizes.Design the mold geometries to allow for at least a six-millimeter border from the edge of the outermost features and the cutting line to permit trimming of the PDMS meniscus following mold casting. This will allow the finished mold to lay flush with the bottom of the culture dish. Next, open the color picker in the top left-hand corner of the window and define new color swatches that are compatible with the laser cutter software.Select each path and assign it an appropriate color from the cut swatch. When finished, assign None for the fill color. Similarly, assign swatch colors to etching paths by selecting the object and then selecting the etching path for the fill color and None for the path color from the color picker.Save designs as either ai or. pdf file formats, depending on which is appropriate for the vector graphics program and laser cutter. Prepare the laser cutter for cutting according to the manufacturer's specification and ensure that sufficient ventilation is used.Then place a 1/4th-inch sheet of acrylic onto the laser bed. Adjust the bed height so that it is properly calibrated to the acrylic. Next, open the design file on the computer connected to the laser cutter and open the Print menu.Ensure that the laser cutter is set as the printer and that Do Not Scale is selected in the Scaling dropdown menu to prevent distortion of the design. Then, click the Print button at the bottom of the Printing dialog. In the laser cutter printing utility, click the setting button to set cut etch parameters for each of the design features.Here, set the power, speed, and pulses per inch parameters for each of the previously chosen colors. Once the settings have been entered, click the large green button in the laser cutter software. This will cause the laser cutter to begin to etch and cut the negative master molds.Finally, use a small brush or compressed air to remove any residual debris from the negative master molds. This will prepare the surface for PDMS casting. Prepare a PDMS casting mix according to the manufacturer's specifications.Degas the prepared PDMS in a vacuum chamber connected to a standard line vacuum lab for one hour or until all bubbles have been eliminated. While the PDMS is being degassed, take the outer edge of the mold with vinyl or masking tape, so the tape extends at least three millimeters above the etched face of the negative master mold. Be sure to press the tape firmly against the side of the mold so prevent leaks later on.Next, pour approximately 0.35 milliliters per centimeter squared of PDMS into the master mold so the PDMS reaches a minimum thickness of 1.5 millimeters. Place the PDMS-coated negative master mold into a vacuum chamber and degas again for one hour or until all bubbles have been eliminated. Then, place the degassed PDMS-coated negative master mold onto a level shelf in a 60 degrees Celsius oven for at least six hours to cure the PDMS.Once cured, use a razor blade to cut the individual molds apart while they are still on the negative master mold. Then, slowly and carefully peel each PDMS mold off of the negative master mold. Trim off any regions with a meniscus from casting, which would prevent the mold from lying flat, as well as any excess material or debris.Finally, autoclave the molds using a standard autoclave cycle to sterilize them for tissue culture. To begin, adhere molds to untreated plastic plates. Prepare the fibrinogen, thrombin, and neutralized collagen solutions and harvest the desired cells as described in the accompanying text protocol.Combine the fibrinogen, neutralized collagen, and cell suspension to create a casting mix. Keep the casting mix on ice. Next, use a handheld high frequency generator to treat the surfaces of the mold that will be exposed to the casting mix to mitigate PDMS hydrophobicity.Expose the surfaces to the plasma treatment to three to five seconds about five minutes before casting the material. Immediately prior to casting the protein cell mixture, add the thrombin to the casting mix and pipette up and down gently without introducing any bubbles. Working quickly, pipette the casting mix into the molds using care to deposit the mix into all corners and crevices of the mold.Avoid ejecting the mix beyond the first stop of the pipette to prevent bubble formation inside the constructs. Then, place the samples into a humidified 37 degrees Celsius incubator for 45 minutes. After 45 minutes, return the constructs to the hood and cover them with cell media before returning to the incubator.Change the cell media every 48 to 72 hours as needed for the cell and construct type. Finally, follow along in the accompanying text protocol to quantity the tissue compaction and tensile properties of the constructs, as well as to prepare various staining techniques and to measure cell alignment. Over time and culture, cellularized constructs compact due to matrix remodeling.This occurs through both reorganization and degradation of the surrounding matrix and is typically associated with an increase in mechanical stiffness. Directed compaction can also induce stress fields within the tissue, which can be manipulated in cellularized constructs to encourage cell alignment. These two images were taken from cardiac tissue constructs that were grown in three millimeter by 17 millimeter rectangular molds with posts at either end.And these two images are cardiac tissue constructs that were grown unconstrained. The two constructs that were constrained were only able to compact in the remaining two directions and led to stress-induced cellular alignment. Once mastered, this technique can be used to design, fabricate, and implement custom tissue molds in less than one day.Following tissue formation, other methods like mechanical testing and histology can be performed to further understand the effects of alignment on cell morphology and tissue function. After watching this video, you should have a good understanding of how to fabricate custom PDMS tissue molds from laser-cut acrylic masters.

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

Research

JoVE Journal

Bioengineering

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 ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

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 ...