This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation under NSF award no. ECS-0335765. CNS is part of Harvard University.

The protocol to generate aligned functional myocardial tissue can be divided into three major parts. The fabrication of the micropatterned master using soft lithography techniques is not considered part of the following protocol but can be made based on established method6.
1. Microcontact Printing of Fibronectin onto PDMS Substrates
<ol>
	<li>Mix Sylgard 184 (Dow Corning) PDMS elastomer at a 10:1 base to curing agent ratio and degas the assembly using a desiccator to eliminate any air bubbles.</li>
	<li>Cure PDMS against a previously fabricated master with 20 &mu;m wide and 2 &mu;m tall ridges, separated by 20 &mu;m spacing for either 2 days at room temperature or 4 hr at 65 &deg;C to obtain microtextured PDMS stamps.</li>
</ol>
* Curing in a desiccator is highly recommended to eliminate any air bubbles.
<ol start="3">
	<li>Spin coat PDMS onto glass cover slips (e.g. 22x22 mm) at 5,000 rpm for 2 min using a Headway Spin Coater to produce thin and even PDMS substrates of 15 &mu;m thickness.</li>
	<li>Clean stamps with 70% Ethanol to remove dust and dirt particles. Let them air dry.</li>
</ol>
* Stamps can be used several times, however, renewing PDMS stamps on a regular basis is recommended due to a surface abrasion over time.
<ol start="5">
	<li>Place stamps in a SPI Plasma-Prep II Plasma Cleaner and process for 1 min at 275 mTorr to render PDMS surfaces hydrophilic.</li>
</ol>
* Oxygen plasma treatment should be watched. Longer treatment times can result in cracking of the elastomer.
<ol start="6">
	<li>Sterilize stamps with 70% Ethanol for 1 min. Dry quickly with compressed air.</li>
</ol>
* Further steps should be performed in a biological safety cabinet to maintain sterility.
<ol start="7">
	<li>Cover stamp surfaces with Fibronectin solution (50 &mu;g/ml in ddH2O). Let it adsorb at least 10 min.</li>
	<li>Shake off Fibronectin solution from stamps and dry quickly with compressed air.</li>
	<li>Establish conformal contact between stamps and PDMS substrates for 2 min and press firmly.</li>
	<li>Rinse micropatterned substrates with ddH2O. Store them in ddH2O for a maximum of 2 weeks at 4 &deg;C unless initially used. Store PDMS stamps in ddH2O.</li>
</ol>
2. Maintenance of Double Transgenic Embryonic Stem Cell Lines
First, prepare the following media for culturing mouse ES cells:
Mouse Embryonic Fibroblast (MEF)-Medium: Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS) ES cell- or Differentiation-grade, 1% Penicillin-Streptomycin (P/S)
Embryonic Stem Cell (ESC)-Medium: DMEM, 15% FBS ES cell-grade, 1% Non-Essential Amino Acids (NEAA), 0.5% P/S, 0.2% Leukemia Inhibitory Factor (LIF), 0.0007% 2-Mercaptoethanol (2-ME)
<ol>
	<li>Coat 6-well plates with sterile 0.1% Gelatin in ddH2O and let it adsorb 15 min at 37 &deg;C.</li>
	<li>Add MEFs from a thawed aliquot to 50 ml PBS in a conical tube and centrifuge them at 1,000 rpm for 5 min. Resuspend MEFs in MEF-medium.</li>
	<li>After adsorption, aspirate excess Gelatin and add MEFs into the wells. Allow MEFs 1 day to attach.</li>
</ol>
* An aliquot of 10 million MEFs suffices for 3 6-well plates.
<ol start="4">
	<li>When MEFs have attached, add ES cells from a thawed aliquot to 50 ml PBS in a conical tube and centrifuge them at 1,000 rpm for 5 min. Resuspend ES cells in ESC-medium and add them to the wells containing MEFs. Culture ES cells in ESC-medium until confluence is reached.</li>
</ol>
* Once ES cells have reached confluence, passaging is required to avoid overgrowth and to maintain ES cell colonies.
<ol start="5">
	<li>Prepare ES cells for passaging. Aspirate ESC-medium and wash once with sterile PBS.</li>
	<li>Aspirate PBS and add 500 &mu;l of Trypsin. Process 3.5 min at 37 &deg;C.</li>
	<li>Pipette the Trypsin solution up and down several times to break down ES cell colonies and neutralize it with 500 &mu;l of ESC-medium.</li>
	<li>Passage ES cells according to your desired ratio, e.g. transfer 33 &mu;l to another well with previously prepared MEFs to passage 1/30. Maintain ES cells in ESC-Medium.</li>
</ol>
* A passage ratio of 1/30 allows ES cells to reach confluence within 3 days. Passage ES cells according to your in vitro differentiation schedule.
3. In vitro Differentiation of Double Transgenic Embryonic Stem Cell Lines and Isolation and Seeding of Cardiac Progenitors
First, prepare the following media required for the in vitro differentiation of ES cells:
Adaptation-Medium: Iscove's Modified Dulbecco's Medium (IMDM), 15% FBS ES cell-grade, 1% NEAA, 0.5% P/S, 0.2% LIF, 0.0007% 2-ME
Differentiation-Medium: IMDM, 15% FBS Differentiation-grade. 1% NEAA, 0.5% P/S, 0.35% of 0.1M Ascorbic Acid, 0.0007% 2-ME
<ol>
	<li>Start in vitro differentiation when ES cells have reached confluence. Coat 10 cm culture dishes with sterile 0.1% Gelatin in ddH2O and let it adsorb 15 min at 37 &deg;C.</li>
</ol>
* Be aware of the duration of the in vitro differentiation process. It takes 8 days until cardiac progenitors can be isolated. Plan your experiments accordingly.
<ol start="2">
	<li>Harvest ES cells as before. After adsorption, aspirate excess Gelatin and add approximately 1/3-1/2 of the ES cell suspension to prepared culture dishes containing Adaptation-medium. Culture cells for 2 days.</li>
</ol>
* Choose the amount of ES cells for adaptation according to your experiments.
<ol start="3">
	<li>Harvest adapted ES cells into a 50 ml conical tube containing PBS using 1. 5 ml of Trypsin. Centrifuge dissociated ES cells at 1,000 rpm for 5 min and resuspend them in Differentiation-medium.</li>
	<li>Make embryoid bodies (EBs) of approximately 1,000 cells per 10 &mu;l drop in 15 cm culture dishes using a multi-channel pipette. Invert the plates and culture EBs as hanging droplets for 2.5 days at 37 &deg;C.</li>
	<li>After 2.5 days, pool EBs of 6-8 15 cm culture dishes into one using Differentiation-medium and culture them for a further 3.5 days at 37 &deg;C.</li>
	<li>After 3.5 days, collect EBs in a conical 50 ml tube and let them sink to the bottom for approximately 5 min. Suck up the supernatant and wash EBs with sterile PBS. Let EBs sink again to the bottom for approximately 5 min.</li>
	<li>Dissociate EBs by processing them with 2. 5 ml of Trypsin for 2 min in a water bath at 37 &deg;C shaking the tube softly. Then, add 2. 5 ml of sterile PBS to the Trypsin solution and pipette up and down with a 10 ml serological pipette approximately 8-10 times to break down cell clusters. Neutralize Trypsin with 10 ml of FACS buffer containing 7.5% FBS ES cell- or Differentiation-grade and 0.01% DAPI in PBS.</li>
	<li>Centrifuge dissociated EBs at 1,000 rpm for 5 min and resuspend them in approximately 1 ml of FACS buffer. Pipette up and down with a 1 ml pipette approximately 8-10 times to break down cell clusters.</li>
</ol>
* Add FACS buffer according to the estimated amount of dissociated EBs. Too highly concentrated cells may clog during FACS and too low concentrated cells may prolong FACS time unnecessarily.
<ol start="9">
	<li>Transfer cells to a sterile round-bottom tube with a 35 &mu;m cell strainer to obtain a single cell suspension.</li>
	<li>Sort differentiated ES cells using a FACSAria Flow Cytometer and obtain purified populations of CVP.</li>
</ol>
*We developed a multi-step gating strategy to isolate highly purified colored cells. In brief, we first gate on Forward Scatter Amplitude (FSC-A) and Side Scatter Amplitude (SSC-A) (Figure 1A). This allows us to separate whole cells from cellular debris. We then gate on FSC-A and Forward Scatter-Width (FSC-W) (Figure 1B). This allows us to isolate cell singlets from doublets and other cellular aggregates. We then gate on SSC-A and Side Scatter-Width (SSC-W) (Figure 1C). This increases the purity of cell singlets. We then gate on DAPI staining to isolate viable cells (DAPI-negative) from non-viable cells (Figure 1D). We then gate on Phycoeryhthrin Amplitude (PE-A) and PE-Cyanine dye 7 Amplitude (PE-Cy7-A) to obtain true red-positive (R+) and true red-negative (R-) cells and to exclude autofluorescent red-negative cells (Figure 1E). R+ and R- cells are then gated separately on Fluorescein isothiocyanate Amplitude (FITC-A) and PE-A to sort true green-positive (G+) and true green-negative (G-) cells within these populations (Figure 1F+1G). This results in 4 populations of cells as follows: R+G+, R+G-, R-G+ and R-G- (Figure 1H).
<ol start="11">
	<li>Passivate micropatterned substrates with 1% Pluronic F-127 in ddH2O for 10 min. Then, wash them 3x with sterile PBS.</li>
</ol>
* Pluronic F-127 is a surfactant that blocks cell adhesion. It supports confinement of cell adhesion to micropatterned area.
<ol start="12">
	<li>Attach PDMS gaskets around the patterned region and press firmly. Then, seed CVP onto Fibronectin micropatterned substrates.</li>
</ol>

<ol>
	<li>Alcon, A., Cagavi Bozkulak, E., Qyang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+functional+heart+tissue+for+myocardial+repair.">Regenerating functional heart tissue for myocardial repair.</a> Cell Mol. Life Sci. , (2012).</li><li>Chien, K. R., Domian, I. J., 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=Cardiogenesis+and+the+complex+biology+of+regenerative+cardiovascular+medicine.">Cardiogenesis and the complex biology of regenerative cardiovascular medicine.</a> Science. 322, 1494-1497 (2008).</li><li>Domian, I. J., 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=Generation+of+functional+ventricular+heart+muscle+from+mouse+ventricular+progenitor+cells.">Generation of functional ventricular heart muscle from mouse ventricular progenitor cells.</a> Science. 326, 426-429 (2009).</li><li>Chan, B. P., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+in+tissue+engineering:+general+approaches+and+tissue-specific+considerations.">Scaffolding in tissue engineering: general approaches and tissue-specific considerations.</a> Eur. Spine J. 17, 467-479 (2008).</li><li>Eschenhagen, T., Eder, A., Vollert, I., Hansen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+aspects+of+cardiac+tissue+engineering.">Physiological aspects of cardiac tissue engineering.</a> Am. J. Physiol Heart Circ Physiol. 303, H133-H143 (2012).</li><li>Feinberg, A. W., 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=Muscular+thin+films+for+building+actuators+and+powering+devices.">Muscular thin films for building actuators and powering devices.</a> Science. 317, 1366-1370 (2007).</li><li>Li, F., Li, B., Wang, Q. M., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape+regulates+collagen+type+I+expression+in+human+tendon+fibroblasts.">Cell shape regulates collagen type I expression in human tendon fibroblasts.</a> Cell Motil. Cytoskeleton. 65, 332-341 (2008).</li><li>Sarkar, S., Dadhania, M., Rourke, P., Desai, T. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+tissue+engineering:+microtextured+scaffold+templates+to+control+organization+of+vascular+smooth+muscle+cells+and+extracellular+matrix.">Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix.</a> Acta Biomater. 1, 93-100 (2005).</li><li>Isenberg, B. C., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+structure+into+engineered+tissues.">Building structure into engineered tissues.</a> Materials Today. 9, 54-60 (2006).</li><li>Athanasiou, K. A., Zhu, C., Lanctot, D. R., Agrawal, C. M., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+biomechanics+in+tissue+engineering+of+bone.">Fundamentals of biomechanics in tissue engineering of bone.</a> Tissue Eng. 6, 361-381 (2000).</li><li>Feinberg, A. W., 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=Controlling+the+contractile+strength+of+engineered+cardiac+muscle+by+hierarchal+tissue+architecture.">Controlling the contractile strength of engineered cardiac muscle by hierarchal tissue architecture.</a> Biomaterials. 33, 5732-5741 (2012).</li><li>Black, L. D., Meyers, J. D., Weinbaum, J. S., Shvelidze, Y. A., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-induced+alignment+augments+twitch+force+in+fibrin+gel-based+engineered+myocardium+via+gap+junction+modification.">Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification.</a> Tissue Eng. Part A. 15, 3099-3108 (2009).</li><li>Kim, D. 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=Nanoscale+cues+regulate+the+structure+and+function+of+macroscopic+cardiac+tissue+constructs.">Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 565-570 (2010).</li><li>Evans, N. D., 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=Substrate+stiffness+affects+early+differentiation+events+in+embryonic+stem+cells.">Substrate stiffness affects early differentiation events in embryonic stem cells.</a> Eur. Cell Mater. 18, 1-14 (2009).</li><li>Tan, J. L., Liu, W., Nelson, C. M., Raghavan, S., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+approach+to+micropattern+cells+on+common+culture+substrates+by+tuning+substrate+wettability.">Simple approach to micropattern cells on common culture substrates by tuning substrate wettability.</a> Tissue Eng. 10, 865-872 (2004).</li><li>Leu, M., Ehler, E., Perriard, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+postnatal+growth+of+the+murine+heart.">Characterisation of postnatal growth of the murine heart.</a> Anat. Embryol. (Berl). 204, 217-224 (2001).</li><li>Li, F., Wang, X., Capasso, J. M., Gerdes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transition+of+cardiac+myocytes+from+hyperplasia+to+hypertrophy+during+postnatal+development.">Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development.</a> J. Mol. Cell Cardiol. 28, 1737-1746 (1996).</li><li>Porrello, E. R., 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=Heritable+pathologic+cardiac+hypertrophy+in+adulthood+is+preceded+by+neonatal+cardiac+growth+restriction.">Heritable pathologic cardiac hypertrophy in adulthood is preceded by neonatal cardiac growth restriction.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, 672-680 (2009).</li><li>Snir, 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=Assessment+of+the+ultrastructural+and+proliferative+properties+of+human+embryonic+stem+cell-derived+cardiomyocytes.">Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes.</a> Am. J. Physiol. Heart Circ. Physiol. 285, H2355-H2363 (2003).</li><li>Ohler, 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=Two-photon+laser+scanning+microscopy+of+the+transverse-axial+tubule+system+in+ventricular+cardiomyocytes+from+failing+and+non-failing+human+hearts.">Two-photon laser scanning microscopy of the transverse-axial tubule system in ventricular cardiomyocytes from failing and non-failing human hearts.</a> Cardiol. Res. Pract. 2009, 802373 (2009).</li><li>Takahashi, H., Nakayama, M., Shimizu, T., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+cell+sheets+for+constructing+three-dimensional+tissue+with+well-organized+cell+orientation.">Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation.</a> Biomaterials. 32, 8830-8838 (2011).</li><li>Williams, C., Xie, A. W., Yamato, M., Okano, T., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stacking+of+aligned+cell+sheets+for+layer-by-layer+control+of+complex+tissue+structure.">Stacking of aligned cell sheets for layer-by-layer control of complex tissue structure.</a> Biomaterials. 32, 5625-5632 (2011).</li></ol>

The authors declare that they have no competing financial interests.

In this protocol, we presented a method to isolate purified populations of cardiogenic progenitors and to seed them on micropatterned Fibronectin substrates what allows them align and take a cardiac myocyte-like rod shape. Normal cellular organization is critical for normal tissue function8,10 , in particular for myocardial tissue. Cardiac myocytes have been shown to develop improved mechanical11,12 and electrophysiological properties11,13 when forming anisotropic cell arrays. Sinc...

Despite recent advances in medical therapy, advanced heart failure remains a leading cause of mortality and morbidity in the developed world. The clinical syndrome arises from a loss of functional myocardial tissue and subsequently an inability of the failing heart to meet the metabolic demands of affected individuals. Since the heart has a limited regenerative capacity, autologous heart transplantation is the only current clinically accepted therapy directly aimed at replenishing lost functional heart tissue. Significant drawbacks of heart transplantation, including a limited number of donor hearts and the need for long-term immunosuppressive therapy, preclude the wide spread use of this therapy. As a result, medical therapy has been the mainstay of treatment for patients with heart disease. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased myocardial physiology in vitro.
The recent convergence of stem cell biology and tissue engineering technology raises new promising avenues for cardiac regeneration. Generating functional myocardial tissue from a renewable patient-specific source would be a major advance in the field. This would allow for the development of disease specific cellular assays for drug development and discovery and would lay the foundation for cardiac regenerative medicine. Human embryonic stem (ES) cells and, more significantly, human induced pluripotent stem (iPS) cells represent a potentially renewable patient-specific source of ventricular progenitor cells and mature ventricular myocytes. The combination of stem cell biology and tissue engineering strategies addresses this problem by generating functional heart tissue in vitro that can be used in the development of cellular assays for drug discovery or in cardiac regenerative approaches for the treatment of advanced heart failure.
A central challenge in cardiac regeneration has been the identification of an optimal cell type. A wide array of cells have been studied1, however to date, although different multipotent cardiogenic precursors from various sources have been discovered, the identification of a cell source that meets critical requirements such as commitment to the myogenic cell fate, maintenance of the capacity to expand in vivo or in vitro and composition to a functional myocardial tissue has proven to be a central problem2. We have previously described a double transgenic murine system that enables the isolation of highly purified populations of committed ventricular progenitors (CVP) from ES cells differentiating in vitro. We generated a novel double transgenic mouse that expresses the red fluorescent protein dsRed under the control of an Isl1-dependent enhancer of the MEF2c gene and the enhanced green fluorescent protein under the control of the cardiac-specific Nkx2.5 enhancer. Based on this two-colored fluorescent reporter system, first and second heart field progenitors from developing ES cells can be isolated by means of Fluorescence Activated Cell Sorting (FACS) according to their expression of MEF2c and Nkx2.5 genes. CVP resemble cardiac myocytes based on the expression patterns of myocardial markers and structural and functional qualities3, what offers great promise for cardiac tissue regenerative purposes.
Although there have been many advances in the field of tissue engineering, mimicking native cellular architecture remains a key challenge. Conventional methods to address this challenge include seeding biological or synthetic scaffolds with cells in vitro. Due to a number of disadvantages of scaffolds, including rapid degradation, limited physical and mechanical stability and low cell density1,4,5 , we have attempted to engineer scaffold-less tissue. Although cardiac cells can modify their local microenvironment by the secretion of extracellular matrix proteins, they have a more limited capacity to organize themselves to rod shaped cardiac myocytes in the absence of extracellular cues. Thus, a template that provides cells appropriate spatial and biological cues to compose functional myocardial tissue is required. Microcontact printing addresses this challenge by providing a simple and inexpensive technique to precisely control cell shape, organization, and function6-8, all of which are crucial for the generation of aligned functional myocardial tissue. It comprises the use of microtextured Polydimethylsiloxane (PDMS) stamps with feature sizes ranging down to 2 &mu;m9 that enable the deposition of extracellular matrix proteins onto PDMS substrates in precise patterns and thus to affect cell adhesion spatially.
Herein, we propose to combine tissue bioengineering technology with stem cell biology to generate anisotropic functional myocardial tissue. Accordingly, we here demonstrate our recently published approach for the following: (1) the generation of micropatterned protein surfaces on PDMS substrates by microcontact printing for the generation of a template for aligned myocardial tissue, (2) the isolation of highly purified cardiac progenitors from ES cells differentiating in vitro, and (3) the combination of both techniques to generate aligned functional myocardial tissue.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>L-Ascorbic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>A4544</td>
 <td>Prepare 0.1M solution in ddH2O</td>
 </tr>
 <tr>
 <td>Desiccator, Vacuum 10 inch</td>
 <td>Nova-Tech International, Inc.</td>
 <td>55206</td>
 <td></td>
 </tr>
 <tr>
 <td>4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
 <td>Life Technologies</td>
 <td>D1306</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium</td>
 <td>Thermo Scientific</td>
 <td>SH30022.01</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Gibco</td>
 <td>14190</td>
 <td></td>
 </tr>
 <tr>
 <td>FACSAria II Flow Cytometer</td>
 <td>BD Biosciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum ES cell-grade</td>
 <td>Gemini Bio-Products</td>
 <td>100-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum Differentiation-grade</td>
 <td>Gemini Bio-Products</td>
 <td>100-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Fibronectin from bovine plasma</td>
 <td>Sigma-Aldrich</td>
 <td>F4759</td>
 <td>Solubilize to 1 mg/ml in ddH2O</td>
 </tr>
 <tr>
 <td>Fisherfinest Premium Cover Glass 22x22mm</td>
 <td>Fisher Scientific</td>
 <td>12-548-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Gelatin from porcine skin</td>
 <td>Sigma-Aldrich</td>
 <td>G1890</td>
 <td>Prepare 0.1% solution in ddH2O</td>
 </tr>
 <tr>
 <td>Headway Spin Coater</td>
 <td>Headway Research, Inc.</td>
 <td>PWM32-PS-CB15</td>
 <td></td>
 </tr>
 <tr>
 <td>Iscove's Modified Dulbecco's Medium</td>
 <td>Thermo Scientific</td>
 <td>SH30228.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukemia Inhibitory Factor</td>
 <td>Self-prepared from LIF-secreting cell lines</td>
 <td></td>
 <td>Prepare 500x stock solution</td>
 </tr>
 <tr>
 <td>MEM Non-Essential Amino Acids</td>
 <td>Gibco</td>
 <td>11140-050</td>
 <td></td>
 </tr>
 <tr>
 <td>2-Mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M6250</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse Embryonic Fibroblasts</td>
 <td>Harvested from day 12-15 mouse embryos</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-127 </td>
 <td>Sigma-Aldrich</td>
 <td>P2443</td>
 <td>Prepare 1% solution in ddH2O </td>
 </tr>
 <tr>
 <td>Round-Bottom Tube with 35 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>SPI Plasma-Prep II Plasma Cleaner </td>
 <td>SPI Supplies</td>
 <td>11005-AB</td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 Silicone Elastomer Kit</td>
 <td>Dow Corning</td>
 <td>184 SIL ELAST KIT 0.5KG</td>
 <td></td>
 </tr>
 <tr>
 <td>0.25% Trypsin-EDTA</td>
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generation of aligned functional myocardial tissue through microcontact printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

FACS-purification of in vitro differentiated ES cells revealed four distinct populations of progenitors (Figure 1). The presence of micropatterned Fibronectin was confirmed by immunofluorescence microscopy that displayed a complete transfer of continuous Fibronectin lines (Figure 2). Plating of FACS-isolated progenitors onto Fibronectin micropatterns resulted in alignment of the R+G+ and part of the R-G+ populations. Although Pl...

Watch this Scientific Journal Video about Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing at JoVE.com

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

generation-aligned-functional-myocardial-tissue-through-microcontact

Research

JoVE Journal

Bioengineering

11.1K ビュー.  Massachusetts General Hospital and Harvard Medical School. 整列心筋組織の生成が臨床的に有用な目的のために、幹細胞生物学の最近の進歩に適応させるための重要な要件です。ここに我々は、細胞の形や機能の正確な制御のためのマイクロコンタクトプリントのアプローチについて説明します。心臓前駆細胞由来の胚性幹細胞の高度に精製された集団を使用して、我々はそれから異方性、機能心筋組織を生成し?...

ビデオ: Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

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. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...