The authors acknowledge the following funding source: the Magic That Matters Fund for Cardiovascular Research.

1. Preparation of Cardiomyocytes
<ol>
	<li>Coat 6-well plates with basement membrane matrix and culture human-induced pluripotent stem cells (hiPSCs) as previously described17.</li>
	<li>Differentiate hiPSCs into hiPSC-CMs using previously described methods18.</li>
	<li>At 16 - 18 d post-differentiation, suspend the cardiomyocytes by rinsing each well with 2 mL of 1x phosphate-buffered saline (PBS) without calcium or magnesium, followed by incubation with 1 mL/well of trypsin or cell dissociation reagent (see Table of Materials) for 5 min at room temperature.</li>
	<li>Neutralize the trypsin or cell dissociation reagent (see Table of Materials) using an equal volume of Roswell Park Memorial Institute (RPMI) cell media supplemented with B-27 (RPMI/B-27 cell media). Pipette up and down to loosen any adhered cells.</li>
	<li>Collect the suspended cardiomyocytes with a 10-mL serological pipette and transfer them to a 50-mL conical tube.</li>
	<li>Centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a cell pellet.</li>
	<li>Resuspend the pellet in 10 mL of RPMI/B-27 cell media.</li>
	<li>Combine 20 &#181;L of cell suspension with an equal amount of 0.4% Trypan blue solution and mix gently.</li>
	<li>Use a manual hemocytometer to count and obtain the concentration and cell viability of the cell suspension.</li>
</ol>
2. Preparation of Fibroblasts
<ol>
	<li>Initiate a culture of a human cardiac fibroblast (HCF) (adult ventricular type) cell line as described previously16.</li>
	<li>Suspend the HCFs by incubating them with an appropriate amount of trypsin or cell dissociation reagent (see Table of Materials) for 5 min at room temperature16. For a T175 flask, 10 mL of trypsin or cell dissociation reagent was used.</li>
	<li>Neutralize the trypsin or cell dissociation reagent (see Table of Materials) using an equal volume of medium, then transfer the sample to a 50-mL conical tube and centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a pellet.</li>
	<li>Resuspend the pellet in 10 mL of fibroblast growth medium (see Table of Materials).</li>
	<li>Combine 20 &#181;L of the HCF cell suspension with an equal amount of 0.4% trypan blue solution and mix gently.</li>
	<li>Use an automated cell counter or manual hemocytometer to count and obtain the concentration and cell viability of the new cell suspension.</li>
</ol>
3. Preparation of Endothelial Cells
<ol>
	<li>Initiate a culture of a human umbilical vein endothelial cell (HUVEC) line as described previously17. Suspend the HUVECs by incubating them with an appropriate amount of trypsin or cell dissociation reagent (see Table of Materials) for 3 min at room temperature17. Neutralize the trypsin or cell dissociation reagent (see Table of Materials) using an equal volume of medium, then transfer to a 50-mL conical tube and centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a pellet. 
	NOTE: For a T175 flask, 10 mL of trypsin or cell dissociation reagent was used.</li>
	<li>Resuspend the pellet in 10 mL of endothelial cell growth medium (see Table of Materials).</li>
	<li>Combine 20 &#181;L of the HUVEC suspension and stain it with an equal amount of 0.4% Trypan blue solution and mix gently.</li>
	<li>Use an automated cell counter or a manual hemocytometer to count and obtain the concentration and cell viability of the cell suspension.</li>
</ol>
4. Creation of Hanging Drop Spheroids
<ol>
	<li>Place the hanging drop device which contains 850 micropores (each with a diameter of 350 &#181;m) into sterile 6-well plates.</li>
	<li>Isolate the three types of cells as described above: hiPSC-CMs, HCFs, and HUVECs. Combine them in a ratio of 70% iPSC-CMs, 15% HCFs, and 15% HUVECs in a 50-mL conical tube with RPMI/B-27 cell media at a concentration of 2,475,000 cells per mL.</li>
	<li>Dispense 4 mL of the cell suspension (2,475,000 cells per mL) to each well of an ultra-low attachment hanging drop system (with a micropore diameter of 350 &#181;m) seated in a 6-well plate.</li>
	<li>Spheroids will spontaneously form within 12 h of dispensing the cell suspension into the hanging drop device. Continue to culture for a total of 72 h (3 d) at 37 &#176;C, 5% CO2, and 95% humidity for the maturation of the spheroids.</li>
	<li>After 72 h of culture, harvest the spheroids through the pores at the bottom of the hanging drop system by placing the device into a dish with medium and swirling it gently to release the spheroids from the hanging drop device.</li>
</ol>
5. 3-D Patch Creation Using the Novel Net Mold
<ol>
	<li>The base, bottom square plate, bottom net, and 10 layered side nets are assembled to create the novel mold with the aid of a pair of sterile forceps. First, stack and align the base, bottom square plate, and bottom net using the corner posts. Then, stack and layer the 10 side nets in alternating directions to create a net of fine stainless-steel prongs.</li>
	<li>Flush the filling base with PBS or medium to reduce the surface tension and, then, transfer the assembled mold onto the filling base.</li>
	<li>Wet the net mold cavity with PBS or medium in the same method. Slowly fill the spheroids into the presupposed cavity of the desired size (2 x 2 x 1 mm, 4 x 4 x 1 mm, or 6 x 6 x 1 mm). 
	Note: Make sure that 120% of the anticipated volume of spheroids is fed into the cavity so that the cell aggregates are in tight connection and stay static.</li>
	<li>Assemble the top net and top square plate onto the corner posts and secure the stoppers and holding tubes to prevent a washout of the spheroids.</li>
	<li>Transfer the filled net mold system to a 6-well plate with 6 mL of RPMI/B-27 cell media or into a sterile container with enough medium to cover the entire assembled mold.</li>
	<li>Incubate the 6-well plate with the filled mold system on a swinging shaker for 7 - 10 days at 3,000 - 4,000 x g. 
	NOTE: The duration of the incubation is decided by the size of the cardiac patches. With larger patches, the incubation time will need to be increased for cardiac patch maturation.</li>
</ol>
6. Removal of the Patch from the Novel Net Mold
<ol>
	<li>Remove the mold system from the media and place it on the sterilized handling mat in a sterile 10-cm dish.</li>
	<li>Remove the holding tube, stoppers, the top square plate, and the top net. Carefully slide out the layered side nets one by one with a pair of sterile forceps. After decannulation, an intact cardiac patch is obtained atop the bottom net.</li>
	<li>Pick up the bottom net with the patch using sterile forceps and transfer the bottom net into a 35-mm dish with 5 mL of RPMI/B27 media. Gently loosen the patch from the bottom net by slowly swirling the net in the dish, or with a sterile cell scraper. After detachment from the bottom net, the cardiac tissue can be cultured with RPMI/B27 media.</li>
	<li>Continue to incubate the free-floating 3-D cardiac patch in the 35-mm dish. Functional synchronous beating can be observed as early as 24 h after the removal from the mold.</li>
	<li>After removing the patch from the net mold, observe that its shape is maintained with integrity and the intensity of synchronous beating increases with time. 
	NOTE: The 3-D net mold-based cardiac patches exhibited electrical integration of component cardiospheres after decannulation. We observed a beating frequency ranging from 60 to 80 beats per minute that persisted for more than 60 days.</li>
</ol>

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The authors have nothing to disclose.

The significance of this method lies in its reproducibility and the effectiveness of the resultant multilayered cardiac tissue. In the field of cardiac tissue engineering, one of the current goals is to identify a method to construct beating, multilayered, and functional 3-D cardiac patches. We report an efficient and reproducible method of creating multilayered cardiac tissues by direct manual seeding of spheroids composed of cardiomyocytes, endothelial cells, and fibroblasts into a novel net mold. The net mold used in this method has a variety of different sizes for the creation of tissues ranging from 2 x 2 x 1 mm to 6 x 6 x 1 mm. The techniques we have described can also be modified for different mold shapes and systems depending on the desired tissue geometry. The described cell concentrations and ratios were optimized previously for hiPSC-CMs, and modifications to a different cell type will require additional evaluation.
The in vitro creation of 3-D cardiac tissues using bioprinter technology has been explored with the hope of developing therapeutic and diagnostic models. However, current bioprinting methods are cumbersome and require significant time and the use of ancillary support structures such as needles or scaffold materials. The presence of these ancillary support structures decreases the diffusion of nutrients and oxygen18. Therefore, central necrosis is a major concern in current 3-D cardiac constructs, due to the lack of capillaries to supply oxygen and nutrients19. Sakaguchi et al. applied the method of sandwiching oriented cell sheets and using vascular bed bioreactors to develop an endothelial network20. In the net mold system presented here, the mold cavity can allow sufficient diffusion of nutrients and oxygen without the requirement of any supporting structures or scaffolds. Furthermore, the net mold-based method is efficient and does not require specialized skills for both the seeding of the spheroids into the mold and the maintenance of the subsequent patch. As such, it is a quick and inexpensive manner to acquire synchronously beating and functional cardiac patches. 
To optimize the spheroid diameter and the number of spheroids used in each hanging drop device, we carried out troubleshooting and modifications regarding the cell types and quantities. For this net mold-based method, the spheroid diameter and the number of spheroids used were of paramount importance to create a functional cardiac tissue of adequate size. We tried different cell concentrations to optimize the best spheroid size for the cardiac tissue creation. Three types of cells were used to create cardiac spheroids by the hanging drop device, which included 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. Under light microscopy, the spontaneously formed spheroids in each micropore were easily observed to be beating after 72 h. We observed that the spheroids' diameter increased with the cell concentration of the hanging drop device. We tried different concentrations of spheroids, from 100 spheroids to 300 spheroids in each device, with 4 mL of RPMI/B27 medium. With numerous trials, we found that the concentration of 2,475,000 cells per mL yielded an optimal diameter of the resultant spheroids. With the optimized diameter of spheroids, we experienced effortless collection and seeding of the spheroids in the stacked net mold, which were important steps to the net mold-based method for multilayer cardiac tissue creation.
The most critical step of the protocol is the optimization of the spheroid size. It is worth mentioning that the spheroids used in this method are smaller than the size of spheroid bodies used in other bioprinting techniques21. Larger spheroids have been found to have central necrosis due to insufficient diffusion of nutrition and oxygen22. With spheroids with a smaller diameter, adequate diffusion of nutrients and oxygen to the center of each spheroid is more easily accomplished, increasing the cellular viability in all areas of the resultant patch. In the protocol presented here, a spheroid diameter of 350 &#181;m allows for improved cell viability and more contact surface area between the spheroids, which we believe contributes to the successful creation of the cardiac tissue using this net mold. Therefore, this is a critical step in the described methodology. To adapt the protocol to other cell types, additional spheroid size optimization experiments are required. 
The limitations of this approach include the inability to precisely control the stacking of the spheroids. This results in areas of non-uniformity of the resulting tissue patches and heterogenous thickness. Moreover, although the actual hands-on time for the seeding of the spheroids is minimal, the creation of large multilayered patches requires an extended culture time to allow for the fusion of the spheroids.
As for future applications, this novel net mold-based method is an efficient and reproducible method of creating 3-D, scaffold-free cardiac tissues with minimal manual effort. The resulting multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This net mold-based method presents a cost-effective and scalable alternative to current bio-fabrication methods of engineered tissues and has the potential to create clinically applicable cardiac tissues for the treatment of heart failure with the goal of replacing ischemic or dysfunctional cardiomyocytes23. Additionally, this method can be utilized to create cardiac tissue for patient-specific screenings of potential novel drug therapies24.

The goal of current cardiac tissue engineering is to develop a therapy to replace or repair the structure and function of injured myocardial tissue1. Methods to create 3-D cardiac tissue models exhibiting the important contractile and electrophysiological properties of native cardiac tissue have been rapidly expanding2,3. A variety of strategies have been explored and used in studies4,5. These methods range from the use of specific synthetic and natural bioactive hydrogels, such as gelatin, collagen, fibrin, and peptides6, to bio-ink deposition technologies2 and bioprinting technologies7. 
It has been shown that scaffold-free methods can produce comparable tissues as biomaterial-based methods, without the drawbacks of incorporating foreign scaffolding material8. Oren Caspi et al. demonstrated that the incorporation of various types of cells enables the generation of highly vascularized human engineered cardiac tissue9. Chin et al. developed a 3-D printing method for cardiac patch creation from spheroids. Resulting patches are composed of cardiomyocytes, fibroblasts, and endothelial cells in a 70:15:15 ratio10. Spheroids have been shown to be effective "building blocks" of scaffold-free cardiac tissue creation, as they are resistant against hypoxia and possess sufficient mechanical integrity for implantation11,12. Previous studies have demonstrated several fabrication methods for spheroid creation, including the use of the hanging drop method, spinner flasks13, microfluidic systems14, and non-adherent culture surfaces uncoated or coated with agarose micro-molds15. In this protocol, we use the hanging drop device, which contains micropores for condensing hundreds of spheroids at one time.
This study presents a novel and efficient scaffold-free method for cardiac tissue creation, which includes manually seeding the spheroids into a square mold cavity and incubating the tissue on a shaker for maturation. Under usual static culture conditions, oxygen diffusion is limited to the outer aspects of the tissue construct, resulting in central necrosis. However, with the net mold, all the spheroids seeded into the mold are immersed in media with a constant fluidic motion, allowing for the increased diffusion of nutrients and oxygen. Additionally, this mold-based method allows for the simultaneous creation of different-sized tissue patches with minimal manual effort and the resultant tissue can be easily removed from the mold. This novel method allows for the efficient and reproducible creation of scaffold-free, multilayered cardiac patches.

<table><tbody><tr><td>Human Cardiac fibroblasts (HCF)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>FM-2 Consists of Basal Medium</td><td>Sciencell</td><td>2331</td><td>HCF culture medium</td></tr><tr><td>Human umbilical vein endothelial cells (HUVEC)</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>EGM+Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC5035</td><td>HUVEC culture medium</td></tr><tr><td>E8 media&amp;nbsp;</td><td>Invitrogen</td><td>A1517001</td><td>HiPSC culture medium</td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Invitrogen</td><td>A1413202</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X)</td><td>Thermo Fisher</td><td>12604013</td><td>Trypsin and Cell dissociation reagent</td></tr><tr><td>RPMI media</td><td>Invitrogen</td><td>11875093</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>B-27 supplement (50x)</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI media with B-27 supplement is hiPSC-CM culture medium</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Novel net mold&amp;nbsp;</td><td>TissueByNet Co.,Ltd</td><td>NM25-1</td><td></td></tr><tr><td>Hanging drop plate</td><td>Kuraray Co.,Ltd</td><td>MPc350</td><td></td></tr><tr><td>6 well plates&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS-3516</td><td></td></tr></tbody></table>

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.

In our experiments, we utilized a cell suspension of 70% iPSC-CMs, 15% HCFs, and 15% HUVECs in RPMI/B-27 cell media at a concentration of 2,475,000 cells per mL. After creating the cell suspension, we dispensed 4 mL of the cell suspension to each well of an ultra-low attachment hanging drop system, as described in step 4.3 of the protocol. The use of the hanging drop system resulted in the spontaneous formation of hundreds of beating spheroids after 3 days of culture at 37 &#176;C, 5% CO2, and 95% humidity. The spheroids were easily observed to be beating under light microscopy at 4X and 10X magnification with an average diameter of 350 &#181;m after 72 h of culture (Figure 1).
At the end of step 6.5 of the protocol, after harvesting the spheroids, the mold was easily seeded with simple pipetting methods. Culturing of the mold allowed for a fusion of the spheroids; subsequent removal of the mold resulted in an intact cardiac patch with mechanical integrity. After removal from the mold and 24 h of culture, functional and synchronous beating of the cardiac tissue was observed, confirming the mechanical integration of the spheroids (Figure 2, left). We also observed that the multiple layers of spheroids were beating spontaneously in a coordinated fashion on light microscopy (Figure 2, right).
An immunohistochemical analysis of the net mold-based 3-D cardiac patch was also performed with the cardiac marker troponin T. Confocal imaging showed that the well-aligned cardiomyocytes all exhibited troponin T expression (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58252/58252fig1.jpg" /> 
Figure 1: Creation of hanging drop spheroids. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) were isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs, at a concentration of 2,475,000 cells per mL. After 72 h, the spontaneously formed spheroids in each micropore were easily observed to be beating under light microscopy. The scale bar in the left panel = 1,000 &#181;m; the scale bar in the right panel = 400 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58252/58252fig2.jpg" /> 
Figure 2: Creation of a multilayered cardiac tissue displaying integration of the component spheroids. After detachment from the net mold, the 3-D cardiac patch maintained its shape and demonstrated synchronous beating, indicating the mechanical integration of the spheroids, as shown in the left panel (where the scale bar = 1,000 &#181;m). The stacked multilayers of spheroids were inspected with light microscopy, as shown in the right panel (where the scale bar = 400 &#181;m), and observed to be beating spontaneously. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58252/58252fig3.jpg" /> 
Figure 3: Immunofluorescence evaluation of the cardiac tissue. On immunofluorescence analysis, the net mold-based 3-D cardiac tissue was found to consist of well-aligned cardiomyocytes with troponin T expression. The scale bar = 50 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58252/58252fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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. ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K Views.  The First Hospital of Jilin University. This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

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

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. ...

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 ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

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 ...

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 ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

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 ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...