Name: Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair (Video) | JoVE Journal
Uploaded: 2020-06-09T13:00:00.000+00:00
Duration: 5 min 5 s
Description: Learn to create mesh-shaped engineered cardiac tissues from human iPS cells for cardiac regenerative therapy. This protocol demonstrates restoration of cardiac function in a rat myocardial infarction model.

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5 minutes

This work was financially supported by the Kosair Charities Pediatric Heart Research Program at the University of Louisville and the Organoid Project at the RIKEN Center for Biosystems Dynamics Research. HiPSCs used in our published protocols were provided by the Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.

1. Maintenance of hiPSCs and cardiovascular differentiation
<ol>
	<li>Expand and maintain hiPSCs on thin-coat basement membrane matrix (growth factor reduced, 1:60 dilution) in conditioned medium extracted from mouse embryonic fibroblasts (MEF-CM) with human basic fibroblast growth factor (hbFGF)4. 
	NOTE: We used a hiPSCs (4-factor (Oct3/4, Sox2, Klf4 and c-Myc) line: 201B6). Add hbFGF at the appropriate concentration for each cell line. Laminin-511 fragment can also be used for the coating of culture dish instead of basement membrane matrix. Commercially available medium designed for feeder-free culture of hiPSCs can be used as a substitute for MEF-CM.</li>
	<li>Use Versene (0.48 mM ethylenediaminetetraacetic acid (EDTA) solution) to detach and dissociate cells when cell confluency reaches 90&#8210;100%. 
	NOTE: Other commercially available products for cell dissociation can also be used.</li>
	<li>Plate cells at a density of 10,000 cells/mm2 on matrix-coated cell culture plates in MEF-CM with hbFGF and culture for two to three days.</li>
	<li>When the culture becomes fully confluent, cover the cells with matrix (1:60 dilution with MEF-CM) for one day.</li>
	<li>Replace the MEF-CM with RPMI 1640 + B27 medium. Add 100 ng/mL of Activin A and 100 ng/mL of Wnt3A to the medium for one day. 
	NOTE: This is day 0 of differentiation. To upregulate canonical Wnt signaling, GSK3&#946; inhibitors can also be used instead of Wnt3A.</li>
	<li>On day 1 of differentiation, change the medium to new RPMI 1640 + B27 with 10 ng/mL of BMP4 and 10 ng/mL of hbFGF. Culture cells for two (MC protocol) or four (CM+EC protocol) days without medium change5. 
	NOTE: CM+EC protocol is optimized to simultaneously induce cardiomyocytes (CMs) and vascular endothelial cells (ECs). MC protocol is optimized to preferentially induce vascular mural cells (MCs).</li>
	<li>CM+EC protocol for the induction of CMs and ECs (Figure 1A).
	<ol>
		<li>Replace the medium on day 5 of differentiation with RPMI 1640 + B27 supplemented with 50 ng/mL of VEGF165.</li>
		<li>Change the culture medium every 48 h until day 13&#8210;15 of differentiation.</li>
	</ol>
	</li>
	<li>MC protocol for the induction of vascular mural cells (Figure 1B)
	<ol>
		<li>Replace the medium with RPMI1640 + 10% fetal bovine serum (FBS) at day 3 of differentiation.</li>
		<li>Change the culture medium every 48 h until day 13&#8210;15 of differentiation.</li>
	</ol>
	</li>
</ol>
2. Cell harvest and lineage analysis on differentiation day 13&#8210;15
<ol>
	<li>Wash the cells with Ca2+ and Mg2+ free phosphate-buffered saline (PBS).</li>
	<li>Add cell dissociation solution (containing proteases, collagenases and DNAses) in the cell culture dish to cover the plate. Incubate the plate for 5 min at 37 &#176;C.</li>
	<li>Collect and dissociate the cells with culture medium using a pipette after incubation.</li>
	<li>Allocate 1 x 106 cells for lineage analysis with flow cytometry. To eliminate dead cells, stain the cells with fixable viability dye.</li>
	<li>Stain the cells with membrane surface markers in PBS with 5% FBS. Use the following dilutions of antibody in fluorescence activated cell sorting (FACS) staining buffer: anti-PDGFR&#946; (1:100), anti-VE cadherin (1:100), anti-TRA-1-60 (1:20).</li>
	<li>For intracellular proteins, resuspend and fix the cells with 4% paraformaldehyde (PFA) in PBS.</li>
	<li>Stain the cells with anti-cardiac isoform of Troponin T (cTnT) in PBS with 5% FBS and 0.75% Saponin, then label the cTnT antibody with 488 mouse IgG1 (dilution 1:50).</li>
	<li>Resuspend the stained cells in PBS with 5% FBS and put them in FACS tubes with cell strainer.</li>
	<li>Analyze the cell composition of the stained cells from each differentiation protocol with flow cytometry to facilitate the generation of cell suspensions with defined lineage distributions. 
	NOTE: While performing this procedure, the remaining cell suspension for ECTs is preserved in a 4 &#176;C refrigerator.</li>
</ol>
3. Fabrication of PDMS tissue mold
<ol>
	<li>Cast a 0.5 mm thick and over 30 mm x 30 mm layer of polydimethylsiloxane (PDMS) by mixing the prepolymer and cross-linking solution at a ratio of 10:1 and then cure at 80 &#176;C for 3 h.</li>
	<li>Cut the PDMS sheet and bond it with silicone adhesive to fabricate a 21 mm x 20.5 mm rectangular tray with 7 mm long, 0.5 mm wide, and 2.5 mm high rectangular posts at a staggered position. Horizontal post spacing between two lines of posts is 2.5 mm (Figure 2B).</li>
	<li>Autoclave the tray at 120 &#176;C for 20 min.</li>
	<li>Coat the tray with 1% poloxamer 407 in PBS for 1 h. Rinse the poloxamer 407 and rinse the mold with PBS sufficiently prior to use.</li>
</ol>
4. ECT construction
<ol>
	<li>After the cell lineage analysis, combine the cells from CM+EC and MC protocols so that the final concentration of MCs is 10 to 20% in a total cell number of six million cells per construct5.</li>
	<li>Suspend mixed six million cells in ECT culture medium (alpha minimum essential medium supplemented with 10% FBS, 50 &#181;M 2-mercaptoethanol and 100 U/mL Penicillin-Streptomycin).</li>
	<li>Preparation of matrix solution
	<ol>
		<li>Mix 133 &#181;L of acid-soluble rat tail collagen type I solution (2 mg/mL, pH 3) with 17 &#181;L of 10x minimum essential medium (MEM). Then mix the solution with 17 &#181;L of alkali buffer (0.2 M NaHCO3, 0.2 M HEPES, and 0.1 M NaOH). 
		NOTE: Collagen must be kept on ice (4 &#176;C). Check buffer color and if the medium does not become pinkish when all solutions are mixed, add additional alkali buffer to the medium. The mixing steps must be mix collagen I + 10x MEM, and then add alkali buffer. DO NOT change this order. Do NOT generate bubbles in the mix.</li>
		<li>Add 67 &#181;L of basement membrane matrix to the neutralized collagen solution. 
		NOTE: The mixed solution must be kept on ice (4 &#176;C).</li>
	</ol>
	</li>
	<li>Centrifuge the prepared cell suspension containing six million cells at 1,100 rpm (240 x g) for 5 min and resuspend the cells with 167 &#181;L of high-glucose DMEM + 20% FBS + 1% penicillin-streptomycin (100x).</li>
	<li>Mix the cell suspension and the matrix solution. The total volume of cell/matrix mixture for one construct is 400 &#956;L. 
	NOTE: The cell/matrix mixture should be non-viscous and a pinkish color at this step. Keep it on ice as the gel will solidify at room temperature. The final concentration of collagen type I is 0.67 mg/mL.</li>
	<li>Pour the cell/matrix mixture evenly over the poloxamer 407 coated PDMS tissue mold, which is placed in a six-well culture plate12. 
	NOTE: Pour the mixture carefully to avoid generating bubbles in order to prevent filling defects in the poured gel.</li>
	<li>Incubate the cell/matrix mixture in a standard CO2 incubator (37C, 5 % CO2) for 60 min.</li>
	<li>After the tissue is formed, soak the tissue mold with 4 mL of ECT culture medium. 
	NOTE: Although the cell/matrix is crosslinked in 60 min, the construct is still very fragile. Add medium gently to avoid damaging it.</li>
	<li>Culture the tissue for 14 days with medium change every day.</li>
	<li>Prior to ECT implantation, remove the ECT from the loading posts gently using sterilized fine forceps. 
	NOTE: The final ECT dimensions after removal from the mold are less than the original mold. A 2.1 cm x 2.05 cm mold generates a released ECT of approximately 1.5 cm x 1.5 cm. A larger 3.9 cm x 4.05 cm mold generates an ECT of approximately 3 cm x 3 cm. Unloaded ECT shows intrinsic spontaneous beating in warm culture medium. Although it initially maintains a mesh structure, an unloaded ECT shrinks and condenses over time. It is possible to hold the tissue softly with fine forceps and then use 7-0 silk suture to attach the ECT to the epicardium.</li>
</ol>

<ol><li>Sanganalmath, S. K., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+therapy+for+heart+failure%3A+A+comprehensive+overview+of+experimental+and+clinical+studies%2C+current+challenges%2C+and+future+directions%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Cell therapy for heart failure: A comprehensive overview of experimental and clinical studies, current challenges, and future directions.</a> Circulation Research. 113, 810-834 (2013).</li>
<li>Fisher, S. A., Doree, C., Mathur, A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meta-Analysis+of+Cell+Therapy+Trials+for+Patients+With+Heart+Failure%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Meta-Analysis of Cell Therapy Trials for Patients With Heart Failure.</a> Circulation Research. 116, 1361-1377 (2015).</li>
<li>Menasché, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+embryonic+stem+cell-derived+cardiac+progenitors+for+severe+heart+failure+treatment%3A+first+clinical+case+report%3A+Figure+1%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report: Figure 1.</a> European Heart Journal. 36, 2011-2017 (2015).</li>
<li>Masumoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+iPS+cell-engineered+cardiac+tissue+sheets+with+cardiomyocytes+and+vascular+cells+for+cardiac+regeneration%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration.</a> Scientific Reports. 4, 6716(2014).</li>
<li>Masumoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myocardial+regenerative+potential+of+three-dimensional+engineered+cardiac+tissues+composed+of+multiple+human+iPS+cell-derived+cardiovascular+cell+lineages%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.</a> Scientific Reports. 6, 29933(2016).</li>
<li>Zimmermann, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature Medicine. 12, 452-458 (2006).</li>
<li>Fujimoto, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+fetal+cardiac+graft+preserves+its+cardiomyocyte+proliferation+within+postinfarcted+myocardium+and+sustains+cardiac+function%5BTitle%5D)+AND+%22Tissue+engineering.+Part+A%22%5BJournal%5D)">Engineered fetal cardiac graft preserves its cardiomyocyte proliferation within postinfarcted myocardium and sustains cardiac function.</a> Tissue engineering. Part A. 17, 585-596 (2011).</li>
<li>Lancaster, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+treatment+for+heart+failure%3A+cell-based+therapy+with+engineered+tissue%5BTitle%5D)+AND+%22Vessel+Plus%22%5BJournal%5D)">Surgical treatment for heart failure: cell-based therapy with engineered tissue.</a> Vessel Plus. 2019, (2019).</li>
<li>Bian, W., Liau, B., Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesoscopic+hydrogel+molding+to+control+the+3D+geometry+of+bioartificial+muscle+tissues%5BTitle%5D)+AND+%22Nature+protocols%22%5BJournal%5D)">Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.</a> Nature protocols. 4, 1522-1534 (2009).</li>
<li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+cardiac+patch+for+advanced+functional+maturation+of+human+ESC-derived+cardiomyocytes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.</a> Biomaterials. 34, 5813-5820 (2013).</li>
<li>Christoforou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cell-derived+cardiac+progenitors+differentiate+to+cardiomyocytes+and+form+biosynthetic+tissues%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.</a> PloS one. 8, 65963(2013).</li>
<li>Nakane, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+Cell+Composition+and+Geometry+on+Human+Induced+Pluripotent+Stem+Cells-Derived+Engineered+Cardiac+Tissue%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue.</a> Scientific Reports. 7, 45641(2017).</li>
<li>Kowalski, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+Cardiomyocyte+Alignment+from+Three-Dimensional+%283D%29+Confocal+Microscopy+of+Engineered+Tissue%5BTitle%5D)+AND+%22Microscopy+and+Microanalysis%22%5BJournal%5D)">Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.</a> Microscopy and Microanalysis. 1, (2017).</li>
</ol>

The authors have no financial or scientific conflicts to disclose.

Following the completion of our investigation of a linear format, hiPSC derived ECT5, we adapted the protocol to mix hiPSC-derived CMs, ECs, and MCs to facilitate the in vitro expansion of vascular cells within ECTs and subsequent in vivo vascular coupling between ECTs and recipient myocardium.
To facilitate the generation of larger, implantable mesh ECT geometries we used thin PDMS sheets to design the 3D molds with loading posts arrayed at staggered positions. During ...

Numerous preclinical studies and clinical trials have confirmed the efficiency of cell-based cardiac regenerative therapies for failing hearts1,2,3. Among various cell types, human induced pluripotent stem cells (hiPSCs) are promising cell sources by virtue of their proliferative ability, potential to generate various cardiovascular lineages4,5, and allogenicity. In addition, tissue engineering technologies have made it possible to transfer millions of cells onto a damaged heart5,6,7,8.
Previously, we reported the generation of three-dimensional (3D) linear engineered cardiac tissues (ECTs) from hiPSC-derived cardiovascular lineages using a commercially available culture system for 3D bioartificial tissues5,7. We found that the coexistence of vascular endothelial cells and mural cells with cardiomyocytes within the ECT facilitated structural and electrophysiological tissue maturation. Furthermore, we validated the therapeutic potential of implanted hiPSC-ECTs in an immune tolerant rat myocardial infarction model to improve cardiac function, regenerate myocardium, and enhance angiogenesis5. However, the linear ECTs constructed by this method were 1 mm by 10 mm cylinders and therefore not suitable for the implantation in preclinical studies with larger animals or clinical use.
Based on the successful use of tissue molds to generate porous engineered tissue formation using rat skeletal myoblasts and cardiomyocytes9, human ESC-derived cardiomyocytes10 and mouse iPSCs11, we developed a protocol to generate scalable hiPSC-derived larger implantable tissue using polydimethylsiloxane (PDMS) molds. We evaluated a range of mold geometries to determine the most effective mold characteristics. Mesh-shaped ECTs with multiple bundles and junctions exhibited excellent characteristics in cell viability, tissue function and scalability compared to plain-sheet or linear formats that lacked pores or junctions. We implanted the mesh-shaped ECT in a rat myocardial infarction model and confirmed its therapeutic effects similar to implanted cylindrical ECTs12. Here we describe the protocol to generate a hiPSC-derived mesh-shaped ECT.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Dishes 100x20 mm style</td><td>Falcon/ Thomas scientific</td><td>9380C51</td><td></td></tr><tr><td>Multiwell Plates For Cell Culture 6well 50/CS</td><td>Falcon / Thomas scientific</td><td>6902A01</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>761036</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accumax</td><td>Innovative Cell Technologies</td><td>AM-105</td><td></td></tr><tr><td>BMP4, recombinant (10&amp;micro;g)</td><td>R&amp;amp;D</td><td>RSD-314-BP-010</td><td></td></tr><tr><td>Collagen, Type I solution from rat tail</td><td>Sigma</td><td>C3867</td><td></td></tr><tr><td>Growth factor-reduced Matrigel</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Human VEGF (165) IS, premium grade</td><td>Miltenyi</td><td>130-109-385</td><td></td></tr><tr><td>Pluronic F-127, 0.2 &amp;micro;m filtered (10% Solution in Water)</td><td>Molecular Probes</td><td>P-6866</td><td></td></tr><tr><td>Recombinant human bFGF</td><td>WAKO</td><td>060-04543</td><td></td></tr><tr><td>Recombinant Human/Mouse/Rat ActivinA (50&amp;micro;g)</td><td>R&amp;amp;D</td><td>338-AC-050</td><td></td></tr><tr><td>rh Wnt-3a (10&amp;micro;g)</td><td>R&amp;amp;D</td><td>5036-WN</td><td></td></tr><tr><td>Versene solution</td><td>Gibco</td><td>15040066</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Culture medium and supplements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x MEM</td><td>Invitrogen</td><td>11430</td><td></td></tr><tr><td>2 Mercaptro Ethanol</td><td>SIGMA</td><td>M6250</td><td></td></tr><tr><td>B27 supplement minus insulin</td><td>Gibco</td><td>A1895601</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Gibco</td><td>11965084</td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>NaHCO3</td><td>Any</td><td></td><td></td></tr><tr><td>PBS 1x</td><td>Gibco</td><td>10010-031</td><td></td></tr><tr><td>Penicillin-Streptomycin (5000 U/mL)</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>RPMI1640 medium</td><td>Gibco</td><td>21870092</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>11900024</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Flowcytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-TRA-1-60, FITC, Clone: TRA-1-60, BD Biosciences</td><td>BD / Fisher</td><td>560380</td><td></td></tr><tr><td>anti-Troponin T, Cardiac Isoform Ab-1, Clone: 13-11, Thermo Scientific Lab Vision</td><td>Fisher</td><td>MS-295-P0</td><td></td></tr><tr><td>BD FACS Clean Solution</td><td>BD</td><td>340345</td><td></td></tr><tr><td>BD FACSFlow Sheath Fluid</td><td>BD</td><td>342003</td><td></td></tr><tr><td>BD FACSRinse Solution</td><td>BD</td><td>340346</td><td></td></tr><tr><td>EDTA</td><td>Any</td><td></td><td></td></tr><tr><td>Falcon Tube with Cell Strainer Cap (Case of 500)</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td><td>Molecular Probes</td><td>L34957</td><td></td></tr><tr><td>PDGFRb; anti-CD140b, R-PE, Clone: 28D4, BD Biosciences</td><td>BD / Fisher</td><td>558821</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47306-50G-F</td><td></td></tr><tr><td>VEcad-FITC; anti-CD144, FITC, Clone: 55-7H1, BD Biosciences</td><td>BD / Fisher</td><td>560411</td><td></td></tr><tr><td>Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit</td><td>Molecular Probes</td><td>Z25002</td><td></td></tr></tbody></table>

preparation of mesh-shaped engineered cardiac tissues derived from human ips cells for in vivo myocardial repair

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

Figure 1A,B shows the schematics of CM+EC and MC protocol. After inducing CMs and ECs from CM+EC protocol and MCs from MC protocol, the cells are mixed adjusting final MC concentrations to represent 10 to 20% of total cells. The 2 cm wide tissue mold is fabricated according to the design drawing from 0.5 mm thick PDMS sheet (Figure 2A,B). Six million of CM+EC+MC cells are combined with collagen I, and matrix and poured onto the ...

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

preparation-mesh-shaped-engineered-cardiac-tissues-derived-from-human

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Learn to create mesh-shaped engineered cardiac tissues from human iPS cells for cardiac regenerative therapy. This protocol demonstrates restoration of cardiac function in a rat myocardial infarction model.

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

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