We would like to thank NSF CAREER Award #1653193, Arizona Biomedical Research Commission (ABRC) New Investigator Award (ADHS18-198872), and the Flinn Foundation Award for providing funding sources for this project. The hiPSC line, SCVI20, was obtained from Joseph C. Wu, MD, PhD at the Stanford Cardiovascular Institute funded by NIH R24 HL117756. The hiPSC line, IMR90-4, was obtained from WiCell Research Institute55,56.

The authors declare that they have no competing financial interests.

The formation of an in vitro human cardiac tissue model with enhanced cell-cell interactions and biomimetic 3D structure is imperative for basic cardiovascular research and corresponding clinical applications1. This outlined protocol explains the development of 3D human anisotropic cardiac tissue within a microfluidic device, using co-culture of stem cell-derived CMs with connective CFs encapsulated within a collagen hydrogel, serving to model the complex cell composition and structure of...

Tissue engineering approaches have been widely explored, in recent years, to accompany in vivo clinical findings in regenerative medicine and disease modeling1,2. Significant emphasis has been particularly placed on in vitro cardiac tissue modeling due to the inherent difficulties in sourcing human primary cardiac tissue and producing physiologically relevant in vitro surrogates, limiting the fundamental understanding of the complex mechanisms of cardiovascular diseases (CVDs)1,3. Traditional models have often involved 2D monolayer culture assays. However, the importance of culturing cardiac cells within a 3D environment to mimic both the native landscape of the myocardium and complex cellular interactions has been extensively characterized4,5. Additionally, most models produced thus far have included a mono-culture of CMs differentiated from stem cells. However, the heart is comprised of multiple cell types6 within a complex 3D architecture7, warranting the critical need to improve the complexity of the tissue composition within 3D in vitro models to better mimic cellular constituents of the native myocardium.
To date, many different approaches have been explored to produce biomimetic 3D models of the myocardium8. These approaches range from experimental setups&#160;that allow for the real-time calculation of generated force, from mono-culture CMs seeded on thin films (deemed muscular thin films (MTFs))9, to co-culture cardiac cells in 3D hydrogel matrices suspended among free-standing cantilevers (deemed engineered heart tissues (EHTs))10. Other approaches have focused on implementing micromolding techniques to mimic myocardial anisotropy, from mono-culture CMs in a 3D hydrogel suspended among protruding microposts in a tissue patch11, to mono-culture CMs seeded among indented microgrooves12,13. There are inherent advantages and disadvantages to each of these methods, therefore, it is pertinent to utilize the technique that aligns with the intended application and the corresponding biological question.
The ability to enhance the maturation of stem cell-derived CMs is essential for the successful in vitro engineering of adult-like myocardial tissue and translation of subsequent findings to clinical interpretations. To this end, methods to mature CMs have been widely explored, both in 2D and 3D14,15,16. For example, electrical stimulation incorporated in EHTs, forced alignment of CMs with surface topography, signaling cues, growth factors from co-culture, and/or 3D hydrogel conditions, etc., all lead to a change in favor of CM maturation in at least one of the following: cell morphology, calcium handling, sarcomeric structure, gene expression, or contractile force.
Of these models, the approaches that utilize microfluidic platforms retain certain advantages in nature, such as control of gradients, limited cell input, and minimal necessary reagents. Furthermore, many biological replicates can be generated at once using microfluidic platforms, serving to better dissect the biological mechanism of interest and increase the experimental sample size in favor of statistical power17,18,19. Additionally, using photolithography in the microfluidic device fabrication process enables the creation of precise features (e.g., topographies) at the micro- and nano-level, which serve as mesoscopic cues to enhance the surrounding cellular structure and macro-level tissue architecture18,20,21,22 for different applications in tissue regeneration and disease modeling.
We previously demonstrated the development of a novel 3D cardiac tissue on-chip model that incorporates surface topography, in the form of innate elliptical microposts, to align hydrogel-encapsulated co-cultured cardiac cells into an interconnected, anisotropic tissue20. After 14 days of culture, the tissues formed within the microfluidic device are more mature in their phenotype, gene expression profile, calcium handling characteristics, and pharmaceutical response when compared to monolayer and 3D isotropic controls23. The protocol described herein outlines the method for creating this 3D co-cultured, aligned (i.e., anisotropic) human cardiac tissue within the microfluidic device using hiPSC-derived CMs. Specifically, we explain the methods to differentiate and purify hiPSCs towards CMs, supplementation of hCFs with CMs to produce an established co-culture population, insertion of the cell population encapsulated within the collagen hydrogel into the microfluidic devices, and subsequent analysis of the 3D constructed tissues through contractile and immunofluorescent assays. The resultant 3D engineered micro-tissues are suitable for various applications, including fundamental biology studies, CVD modeling, and pharmaceutical testing.

<table><tbody><tr><td>0.65 mL centrifuge tubes</td><td>VWR</td><td>87003-290</td><td></td></tr><tr><td>1 mm Biopsy punch</td><td>VWR</td><td>95039-090</td><td></td></tr><tr><td>1.5 mm Biopsy punch</td><td>VWR</td><td>95039-088</td><td></td></tr><tr><td>15 mL Falcon tubes</td><td>VWR</td><td>89039-670</td><td></td></tr><tr><td>18x18mm coverslips</td><td>VWR</td><td>16004-308</td><td>The coverslips should be No.1, to allow for high magnification imaging</td></tr><tr><td>4% paraformaldehyde</td><td>ThermoFisher</td><td>101176-014</td><td></td></tr><tr><td>6-well flat botttom tissue-culture plates</td><td>VWR</td><td>82050-844</td><td></td></tr><tr><td>B27 minus insulin</td><td>LifeTech</td><td>A1895602</td><td></td></tr><tr><td>B27 plus insulin</td><td>LifeTech</td><td>17504001</td><td></td></tr><tr><td>CHIR99021</td><td>VWR</td><td>10188-030</td><td></td></tr><tr><td>Collagen I, rat tail</td><td>Corning</td><td>47747-218</td><td></td></tr><tr><td>DMEM F12</td><td>ThermoFisher</td><td>11330057</td><td></td></tr><tr><td>DPBS</td><td>ThermoFisher</td><td>21600069</td><td></td></tr><tr><td>E8</td><td>ThermoFisher</td><td>A1517001</td><td>can also be made in house</td></tr><tr><td>EDTA</td><td>VWR</td><td>45001-122</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>FGM3</td><td>VWR</td><td>10172-048</td><td></td></tr><tr><td>GFR-Matrigel</td><td>VWR</td><td>47743-718</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898-500G</td><td></td></tr><tr><td>Goat serum</td><td>VWR</td><td>10152-212</td><td></td></tr><tr><td>hESC-Matrigel</td><td>Corning</td><td>BD354277</td><td></td></tr><tr><td>IPA</td><td></td><td></td><td></td></tr><tr><td>IWP2</td><td>Sigma</td><td>I0536-5MG</td><td></td></tr><tr><td>Kimwipes</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>MTCS</td><td>Sigma</td><td>440299-1L</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S2002-25G</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>S5881-500G</td><td></td></tr><tr><td>Pen/Strep</td><td>VWR</td><td>15140122</td><td></td></tr><tr><td>Petri dish (150x15mm)</td><td>VWR</td><td>25384-326</td><td></td></tr><tr><td>Petri dish (60x15mm)</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>P3532-5G</td><td></td></tr><tr><td>RPMI 1640</td><td>ThermoFisher</td><td>MT10040CM</td><td></td></tr><tr><td>RPMI 1640 minus glucose</td><td>VWR</td><td>45001-110</td><td></td></tr><tr><td>Silicon Wafers (100mm)</td><td>University Wafer</td><td>1196</td><td></td></tr><tr><td>Sodium lactate</td><td>Sigma</td><td>L4263-100ML</td><td></td></tr><tr><td>SU8 2075</td><td>Microchem</td><td>Y111074 0500L1GL</td><td></td></tr><tr><td>SU8 Developer</td><td>ThermoFisher</td><td>NC9901158</td><td></td></tr><tr><td>Sylgard Elastomer</td><td>Essex Brownell</td><td>DC-184-1.1</td><td></td></tr><tr><td>T75 flasks</td><td>VWR</td><td>82050-856</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-100ML</td><td></td></tr><tr><td>TrypLE</td><td>ThermoFisher</td><td>12604021</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>ThermoFisher</td><td>15400054</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>P9416-50ML</td><td></td></tr><tr><td>Y-27632</td><td>Stem Cell Technologies</td><td>72304</td><td></td></tr><tr><td>EVG620 Aligner</td><td>EVG</td><td></td><td></td></tr><tr><td>Plasma cleaner PDC-32G</td><td>Harrick Plasma</td><td></td><td></td></tr><tr><td>Zeiss AxioObserver Z1 microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Leica SP8 Confocal microscope</td><td>Leica</td><td></td><td></td></tr></tbody></table>

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.

To obtain a highly purified population of CMs from hiPSCs, a modified version involving a combination of the Lian differentiation protocol33 and Tohyama purification steps34 is used (refer to Figure 1A for experimental timeline). The hiPSCs need to be colony-like, ~85% confluent, and evenly spread throughout the culture well 3-4 days after passage, at the onset of CM differentiation (Figure 1B). Specifically, on Da...

Watch this Scientific Journal Video about Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform at JoVE.com

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

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 ...

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Research

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Bioengineering

4.9K Views.  Arizona State University. 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.

Video: Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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. ...

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.

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 ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Biochemistry

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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. ...

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 ...

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 ...