This work was supported by NIH (1F30HL118923-01A1) to T.J.C., NIH/NHLBI PEN contract HHSN268201000045C to K.D.C., the research grant council of Hong Kong TRS T13-706/11(K.D.C), NIH (R01 HL113499) to B.D.G., the American Heart Association (12PRE12060254) to R.J., and Research Grant Council of HKSAR (TBRS, T13-706/11) to R.L. Additional funding was provided to T.J.C. by NIH DRB 5T32GM008553-18 and as a traineeship on NIDCR-Interdisciplinary Training in Systems and Developmental Biology and Birth Defects T32HD075735. The authors also wish to gratefully acknowledge Arthur Autz at The Zahn Center of The City College of New York for assistance with machining the bioreactor and Mamdouh Eldaly for technical assistance. We also thank Dr. Kenneth Boheler for advice on cardiac differentiation, and Dr. Joshua Hare for generously providing human mesenchymal stem cells.

<ol>
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The authors declare that they have no competing financial interests.

Construction of defined human engineered cardiac tissues (hECT) can provide a more consistent and reliable model of human cardiac myocyte function. Critically, all cellular and extracellular components in the system are known and can be manipulated as desired, thus removing the confounding influence of other unknown cell types resulting from the differentiation process. To balance rapid cell growth and high yield, it is preferable that the differentiation starts at 75% confluence of the hESCs, ideally four days after pla...

Cardiac tissue engineering has advanced greatly in the last decade, with multiple groups publishing results of fully functional, beating tissues made from both murine cardiomyocytes1-6 and, more recently, human stem cell-derived cardiac myocytes7-12. The cardiac tissue engineering field is driven by two primary and essentially independent goals: 1) to develop exogenous grafts that can be transplanted into failing hearts to improve function4-6; and 2) to develop in vitro models for studying physiology and disease, or as screening tools for therapeutic development2,7.
Three-dimensional (3-D) cell culture is considered essential for developing next generation screening tools, as the 3-D matrix reflects a more natural cardiac microenvironment than traditional 2-D monolayer cell culture; indeed some aspects of cell biology are fundamentally different in 2-D vs. 3-D cultures13,14. Additionally, engineered cardiac tissues are constructed from completely defined components: an extracellular matrix, and a cell population. For traditional engineered human cardiac tissues, while the extracellular matrix composition (usually fibrin9 or collagen7,8,10) is strictly controlled, the input cell composition is less well defined, with the entire mixture of cells from a directed cardiac differentiation of either embryonic stem cells (ESC7,9) or induced pluripotent stem cells (iPSC10,12) being added to the tissues. Depending on the specific cell line and the efficiency of the differentiation protocol used, the resulting percentage of cardiomyocytes can range from less than 25% to over 90%, the specific cardiomyocyte phenotype (i.e., ventricular-, atrial-, or pacemaker-like) can also vary, even the non-cardiomyocyte fraction can be highly heterogeneous15,16 and alter the maturity of the differentiated cardiac myocytes17.
Recent cardiac tissue engineering work has attempted to control the input population of cells, with either a cardiac reporter human embryonic stem cell line8 or cell surface markers18 being used to isolate the cardiac myocyte component of the differentiation. While initially a tissue composed of only cardiac myocytes would seem to be the ideal, this is in fact not the case; hECTs composed solely of cardiac myocytes fail to compact into functional tissues, with some groups finding a 3:1 ratio of cardiac myocytes:fibroblasts producing the highest twitch force8. By using various cell selection methods, including surface markers for live cell sorting, it is possible to create hECTs with defined cell populations. While markers of non-cardiac stromal cells have been available for some time, such as the putative fibroblast marker CD9019,20, surface markers of cardiac myocytes have been more difficult to identify. SIRP&#945; was among the first cardiac surface markers identified for human cardiac myocytes18 and has been shown to be highly selective for the cardiac lineage. Recently, we have found that double-sorting for SIRP&#945;+ and CD90- cells yields nearly pure cardiomyocytes, with the CD90+ population exhibiting a fibroblast-like phenotype (Josowitz, unpublished observations). Based on these collected findings, herein we describe creating hECTs using a 3:1 combination of SIRP&#945;+/CD90- cardiomyocytes and CD90+ fibroblasts.
The ability to engineer a completely defined human cardiac tissue is essential not only for creating robust screening tools, but also for developing model systems to investigate emerging cell- and gene-based cardiac therapies. In particular, numerous cell therapies for heart failure, utilizing cell types including mesenchymal stem cells (MSC)21, cardiac stem cells22 and bone marrow mononuclear cells23-25, have been tested in clinical trials. While many of the initial results have been promising21,23,25, the initial benefit often diminishes over time26-29. A similar trend has been reported in murine engineered cardiac tissues, which display a significant functional benefit due to MSC supplementation, but the benefit is not sustained during long-term culture1. Underlying the sub-optimal performance is our limited knowledge of the mechanisms governing cell therapies. A deeper understanding of how therapeutic cells exert their beneficial influence, as well as potential negative consequences of myocyte-nonmyocyte interactions, would enable the development of improved therapies yielding clinically significant and sustained benefits, with minimal side effects, for patients with heart failure.
Here, we describe the use of defined hECTs to interrogate mechanisms of cell-based therapy. The controlled tissue composition is essential to identify specific factors impacting cardiomyocyte performance. Directly supplementing hECTs with the therapeutic cell type of interest (e.g., MSCs), can reveal the effects on cardiac myocyte performance, as we have demonstrated in rat ECTs1.
The following multi-step protocol begins with directed cardiac stem cell differentiation, followed by fabrication of the multi-tissue bioreactor, and concluding with a description of tissue construction and functional analysis. Our experiments are performed using the NIH-approved H7 human embryonic stem cell (hESC) line. However, the following protocols have also been tested using an additional hESC line and three induced pluripotent stem cell (hiPSC) lines with similar results. We have found that efficiency in cardiomyocyte differentiation and success in hECT fabrication can be cell line dependent, particularly for hiPSC lines derived from individual patients. By following this protocol, two 6-well dishes are plated with a total of 1.68 million hESCs (140,000 cells per well), which yields approximately 2.5 million myocytes after differentiating for 20 days and sorting, enough to make six defined tissues.

<table><tbody><tr><td>&lt;strong&gt;Cell Culture&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2411</td><td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td></tr><tr><td>B27 with Insulin</td><td>Life Technologies</td><td>17505055</td><td></td></tr><tr><td>B27 without Insulin</td><td>Life Technologies</td><td>A1895601</td><td></td></tr><tr><td>CHIR99021</td><td>Stemgent</td><td>04-0004</td><td>Create 6 &amp;mu;M stock, then aliquot and store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Essential 8 Media</td><td>Life Technologies</td><td>A1517001</td><td></td></tr><tr><td>H7 Human Embryonic Stem Cells</td><td>WiCell</td><td>WA07</td><td></td></tr><tr><td>hESC Qualified Matrix, Corning Matrigel</td><td>Corning</td><td>354277</td><td>Thaw on ice at 4 &amp;deg;C overnight then aliquot 150 &amp;mu;l into separate tubes and store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>IWR-1</td><td>Sigma-Aldrich</td><td>I0161</td><td>Create 10 mM stock and aliquot. Store at -20 &amp;deg;C</td></tr><tr><td>Neonatal Calf Serum</td><td>Life Technologies</td><td>16010159</td><td></td></tr><tr><td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td><td>Stem Cell Technologies</td><td>7174</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875-093</td><td>Keep refrigerated</td></tr><tr><td>Y-27632 (ROCK Inhibitor)</td><td>Stemgent</td><td>04-0012</td><td>Resuspend to a 10 mM stock concentration, aliquot and store at -20&amp;nbsp;&amp;deg;C. Avoid freeze thaw cycles.</td></tr><tr><td>&lt;strong&gt;Cell Sorting&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>Life Technologies</td><td>D1306</td><td></td></tr><tr><td>CD90-FITC</td><td>BioLegend</td><td>328107</td><td></td></tr><tr><td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&amp;nbsp;</td><td>PromoCell</td><td>C-41200</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologics</td><td>S11250</td><td></td></tr><tr><td>SIRP&amp;alpha;-PE/Cy7</td><td>BioLegend</td><td>323807</td><td></td></tr><tr><td>&lt;strong&gt;Tissue Construction&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>0.25% Trypsin/0.1% EDTA</td><td>Fisher Scientific</td><td>25-053-CI</td><td>Optional: For collection of supplemental cells of interest</td></tr><tr><td>10x MEM</td><td>Sigma-Aldrich</td><td>M0275-100ML</td><td></td></tr><tr><td>10x PBS Packets</td><td>Sigma-Aldrich</td><td>P3813</td><td></td></tr><tr><td>Collagen, Bovine Type I</td><td>Life Technologies</td><td>A10644-01</td><td>Keep on ice</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11330057</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagles Medium (DMEM), High Glucose</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Sodium HEPES</td><td>Sigma-Aldrich</td><td>H3784</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisher Scientific</td><td>NC0536757</td><td></td></tr><tr><td>15 ml polyproylene centrifuge tube</td><td>Corning</td><td>352096</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>Corning</td><td>352235</td><td>With integrated 35 &amp;mu;m cell strainer</td></tr><tr><td>50 ml polyproylene centrifuge tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>6-well flat bottom tissue-culture treated plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Cell Scraper, Disposable</td><td>Biologix</td><td>70-2180</td><td></td></tr><tr><td>Polysulfone</td><td>McMaster-Carr</td><td></td><td></td></tr><tr><td>Polytetrafluoroethylene (Teflon)</td><td>McMaster-Carr</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Dissecting Microscope</td><td>Olympus</td><td>SZ-61</td><td>Or similar, must have a mount for the high speed camera to attach</td></tr><tr><td>Electrical Pacing System</td><td>Astro-Med, Inc</td><td>Grass S88X Stimulator</td><td></td></tr><tr><td>High Speed Camera</td><td>Pixelink</td><td>PL-B741U</td><td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td></tr><tr><td>Plate Temperature Control</td><td></td><td></td><td>Used to maintain media temperature during data acqusition.</td></tr><tr><td>&lt;strong&gt;Custom Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>LabView Post-tracking Program</td><td></td><td></td><td>available upon request from the authors</td></tr></tbody></table>

construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

To obtain cardiac myocytes, a slightly modified version of the Boheler and Lian differentiation methods is used30,31. It is imperative that the differentiation starts during the log-phase of cell growth, but also that the starting population is sufficiently confluent to obtain a useable number of cells after sorting (approximately 75% is optimal). Typically, for H7 hESCs, plating at a density of 140,000 hESCs per well of a 6-well dish in essential 8 media and 5% CO2 incubator maintained at 37 &#176;...

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Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

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9.8K Views.  Icahn School of Medicine at Mount Sinai. This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Video: Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

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.

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. ...

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

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

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

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

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

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.

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 ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

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