The authors acknowledge the following funding sources: Magic That Matters Fund for Cardiovascular Research and the Maryland Stem Cell Research Fund (2016-MSCRFI-2735).

1. Preparation of Cardiomyocytes
<ol>
	<li>Generate and culture human induced pluripotent stem cells (hiPSCs) on 6-well plates coated with basement membrane matrix as described10.</li>
	<li>Differentiate hiPSCs into hiPSC-derived cardiomyocytes (hiPSC-CMs) using previously described methods11,12.</li>
	<li>On Day 19 after differentiation, isolate the cardiomyocytes using 2 mL of Trypsin/EDTA 0.05% in each well for 5 min at room temperature.</li>
	<li>Monitor the cardiomyocytes under an optical microscope to watch for cell dissociation.</li>
	<li>Neutralize Trypsin using 2 mL of Trypsin inhibitor 0.0125%.</li>
	<li>Pool the isolated cardiomyocytes and transfer them into one 50 mL conical tube using a motorized pipette filler.</li>
	<li>Centrifuge the cell suspension for 5 min at 250 x g at room temperature to obtain a pellet.</li>
	<li>Resuspend the pellet in 5 mL of Roswell Park Memorial Institute (RPMI) cell media supplemented with B-27 (RPMI/B-27 cell media).</li>
	<li>Pipette 20 &#181;L of cells in cell media and stain with an equal amount of 0.4% Trypan Blue solution.</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>
2. Preparation of Fibroblasts
<ol>
	<li>Initiate the human cardiac fibroblast (HCF) (adult ventricular type) cell line13.</li>
	<li>Passage and isolate them in accordance to HCF culturing protocols13.</li>
	<li>Pipette 20 &#181;L of cells in cell media and stain with an equal amount of Trypan Blue solution 0.4%.</li>
	<li>Use an automated cell counter or a 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 the human umbilical vein endothelial cell (HUVEC) line as described14. Passage and isolate them in accordance to HUVEC culturing protocols as described14.</li>
	<li>Pipette 20 &#181;L of cells in cell media and stain with an equal amount of 0.4% Trypan Blue solution.</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. Co-culture:
<ol>
	<li>Mix the three cell types (hiPSC-CM, HCFs, and HUVECs) in RPMI/B-27 cell media to generate a stock of mixed cell solution in a 50 mL conical tube, at a hiPSC-CM:HCF:HUVEC cell ratio of 70:15:15 and concentration of 165,000 cells per mL.</li>
	<li>Distribute the mixed cell solution into ultra-low attachment 96-well U-bottom plates at 200 &#181;L per well, or 33,000 cells per well, using a multi-channel pipette.</li>
	<li>Incubate the 96-well plates for 3 days (37&#176;C, 5% carbon dioxide, 95% humidity).</li>
	<li>Inspect for the presence of mixed cell multicellular spheroids at the center and bottom of individual wells, under light microscopy. 
	NOTE: In a two-dimensional microscopic projection, these spheroids will appear circular in shape (Figure 1). Spheroids should form within 24 h, after cell seeding into ultra-low attachment 96-well plates. Spheroids should start beating within 48 h, after cell seeding into 96-well plates. Spheroids that are not beating should not be used for 3D bioprinting to ensure that the final 3D bioprinted cardiac patch is functional. Immunofluorescence of spheroids for connexin 43 (Cx43) and troponin may be used to assess spheroid cellular composition and quality15.</li>
</ol>
5. 3D Bioprinting of Scaffold-free Cardiac Tissues
<ol>
	<li>Load the plates containing the spheroids into the magazines of the scaffold-free spheroid-based 3D bioprinter.</li>
	<li>Turn on the 3D bioprinter and execute the 3D bioprinting software.
	<ol>
		<li>Click &#34;Auxillary&#34;, &#34;Operation Preparation&#34;, &#34;Inspect Mode&#34;, and then &#34;Home Return&#34;.</li>
		<li>Click the green icon under &#34;Start&#34; to begin spheroid inspection.</li>
		<li>Watch for the spheroid diameters to appear under the column &#34;(2) Dia (&#181;m)&#34; on the screen.</li>
	</ol>
	</li>
	<li>To estimate the average spheroid diameter to the nearest 100 &#181;m, click &#34;Initial settings&#34; and enter the average spheroid diameter into Stack Z pitch (&#181;m).</li>
	<li>Click &#34;Total&#34;, the number under &#34;Layers&#34; should readjust automatically. Click &#34;Apply&#34; to confirm the settings.</li>
	<li>Click &#34;Needle Array Map&#34;, go to &#34;Layer No.&#34;, select the desired layer for 3D bioprinting, and draw the desired 3D bioprinting design on the map on the left of the screen by selecting individual spheroid positions. Click &#34;Apply&#34; to confirm the 3D bioprinting design.</li>
	<li>Click &#34;Needle Array Check&#34; to begin needle array inspection. If the needle array is out of focus, input values into &#34;OffsetZ (&#181;m)&#34;, ranging from 0 to 500 &#181;m to adjust camera focus, starting with 500 &#181;m and decreasing by 100 &#181;m to 0 &#181;m.</li>
	<li>Click &#34;Inspection Parameters&#34;, &#34;Type 1&#34;, and set &#34;Diameter (&#181;m)&#34; to the desired diameter range. Set &#34;Roundness (%)&#34; and &#34;Smoothness (%)&#34; to desired percentages. Click &#34;Apply&#34; to confirm the settings. 
	NOTE: Here the spheroid diameters were set from 450 &#181;m to 550 &#181;m. &#34;Roundness (%)&#34; and &#34;Smoothness (%)&#34; are computer calculated assessment of each individual spheroid. Here, the settings used for &#34;Roundness (%)&#34; is 60% and &#34;Smoothness (%)&#34; is 70%.</li>
	<li>Click &#34;Presence&#34; and update the &#34;Diameter (&#181;m)&#34; to the same settings in the previous step. Click &#34;Apply&#34; to confirm the settings.</li>
	<li>Click &#34;Stack Mode&#34;, then the green icon under &#34;Start&#34; to begin spheroid pick up using the nozzle of the 3D bioprinter by vacuum suction, and placement of spheroids in specific locations in a needle array. 
	NOTE: For a cardiac patch consisting of 81 spheroids (9 spheroids x 9 spheroids), 3D bioprinting should take about 20 to 30 min.</li>
	<li>After bioprinting, using sterile forceps, pick up the needle array containing the 3D bioprinted patch and transfer it into a 250 mL sterile beaker containing 150 mL of RPMI/B-27 cell media.</li>
	<li>Incubate the 3D bioprinted patch with the needle array for 3 days (37&#176;C, 5% carbon dioxide, 95% humidity).</li>
</ol>
6. Removal of 3D Bioprinted Patch from the Needle Array and Patch Maturation
<ol>
	<li>After 3 days of incubation, with one hand at the base of the needle array and the other on the plastic cover under the patch, gently slide the plastic cover upwards to remove the 3D bioprinted patch from the needle array. 
	NOTE: Lubrication with phosphate-buffered saline (PBS) may be applied to the needles just before this step, as needed, for smoother and easier removal.</li>
	<li>Using a pair of sterile forceps, pick up the plastic cover with the 3D bioprinted patch on top of it and transfer the patch and plastic cover into a 35 mm dish containing RPMI/B-27 cell media.</li>
	<li>Gently shake the plastic cover with the patch in the RPMI/B-27 cell media, with the forceps still holding on to the plastic cover, to release the patch into the media.</li>
	<li>Inspect the 3D bioprinted patch under light microscopy to visualize whether it is beating to ensure that the 3D bioprinted patch is functional.</li>
	<li>Incubate the 3D bioprinted patch in the 35 mm dish for 3 days or longer (37&#176;C, 5% carbon dioxide, 95% humidity). 
	NOTE: After 3 days, the spheroids fuse together and the holes in the patch, where the needles were, should no longer be visible.</li>
</ol>

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I., Hibino, N., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+the+Kenzan+Method+for+Robotic+Cell+Spheroid-Based+Three-Dimensional+Bioprinting.">Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting.</a> Tissue Eng Part B Rev. , (2017).</li><li>Cui, X., Boland, T., D'Lima, D. D., Lotz, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+inkjet+printing+in+tissue+engineering+and+regenerative+medicine.">Thermal inkjet printing in tissue engineering and regenerative medicine.</a> Recent Pat Drug Deliv Formul. 6, 149-155 (2012).</li><li>Murphy, C. M., Haugh, M. G., O'Brien, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+mean+pore+size+on+cell+attachment,+proliferation+and+migration+in+collagen-glycosaminoglycan+scaffolds+for+bone+tissue+engineering.">The effect of mean pore size on cell attachment, proliferation and migration in collagen-glycosaminoglycan scaffolds for bone tissue engineering.</a> Biomater. 31, 461-466 (2010).</li><li>Noguchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+three-dimensional+pre-vascularized+scaffold-free+contractile+cardiac+patch+for+treating+heart+disease.">Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease.</a> J Heart Lung Transpl. 35, 137-145 (2016).</li><li>Mironov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+tissue+spheroids+as+building+blocks.">Organ printing: tissue spheroids as building blocks.</a> Biomater. 30, 2164-2174 (2009).</li><li>Itoh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-Free+Tubular+Tissues+Created+by+a+Bio-3D+Printer+Undergo+Remodeling+and+Endothelialization+when+Implanted+in+Rat+Aortae.">Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae.</a> PloS One. 10, e0136681 (2015).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol Biol. 997, 45-56 (2013).</li><li>Li, S., Cheng, H., Tomaselli, G. F., Li, R. 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The authors have nothing to disclose.

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histological and mechanical properties.
This approach also allows different types of spheroids to be used to make a patch, as well as the 3D bioprinting of cardiac patches with complex 3D designs. This complexity of the final 3D construct is achievable by 3D bioprinting with precise spheroid positioning, but may be difficult to achieve by gentle rotation of floating spheroids7 or simple molding of spheroids into a cylindrical 3D construct16.
The limitations of this approach are the relatively fixed size of the spheroids between 450 to 550 &#181;m due to the fixed distance of 400 &#181;m between the needles in the needle array, as well as possibly reduced cell viability if the spheroids are too large in diameter. The patches are mechanically fragile immediately after removal from the need array; however, the strength and durability are improved with culture for longer durations of time.
Finally, 3D bioprinting of cardiac patches has promising clinical applications17, in cardiac regeneration18,19 and as 3D models of heart disease. These 3D models can be used for applications in predictive pharmacology/toxicology and development of cell and genome directed therapies. 
In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications in cardiac tissue regeneration and as 3D models of heart disease.

There are many different methods of 3D bioprinting1,2,3. 3D bioprinting is frequently classified by printing technology1, with examples such as inkjet bioprinting, microextrusion bioprinting, laser assisted bioprinting, a combination of methods, or newer approaches. 3D bioprinting can also be classified into scaffold-free or scaffold-dependent methods4. Most methods of 3D bioprinting are scaffold-dependent, where there is a need for biomaterials, e.g. bioinks5 or scaffolds6. However, scaffold-dependent 3D bioprinting face many issues and limitations4,7, such as immunogenicity of scaffolding material, cost of proprietary bioinks, slow speed and toxicity of degradation products.
Scaffold-free cardiac tissue engineering using spheroids has been attempted8, with the potential to overcome these disadvantages of scaffold-dependent tissue engineering. However, as acknowledged by the authors in that paper, it had been difficult to robustly handle and position spheroids in fixed locations, in the process of biofabrication. The concomitant use of 3D bioprinting and spheroid-based tissue engineering has the potential to overcome these difficulties. In this protocol, we describe 3D bioprinting of cardiac tissue without other biomaterials, using only cells in the form of spheroids.
Scaffold-free spheroid-based 3D bioprinters9 have the ability to pick up individual spheroids using vacuum suction and position them on a needle array. The concept of positioning spheroids on a needle array in 3D bioprinting, is inspired from the use of needle arrays (known as &#34;kenzan&#34;) in the ancient Japanese art of flower arrangement, ikebana. This system allows spheroids to be precisely positioned in any configuration and results in the individual spheroids fusing together over a short period to create a 3D bioprinted tissue. This method thus allows spheroids to be manipulated with ease, with potential implications for the future of scaffold-free organ biofabrication.

<table><tbody><tr><td>Geltrex</td><td>Invitrogen&amp;nbsp;</td><td>A1413202</td><td></td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Thermo Fisher</td><td>15400054</td><td></td></tr><tr><td>Defined Trypsin inhibitor 0.0125%</td><td>Thermo Fisher</td><td>R007100</td><td></td></tr><tr><td>RPMI Cell Media</td><td>Invitrogen</td><td>11875-093</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>B-27 Supplement</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Human cardiac fibroblasts (adult ventricular type)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>Human umbilical vein endothelial cells</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>PrimeSurface ultra-low attachment 96-well U-bottom plates&amp;nbsp;</td><td>Akita Sumitomo Bakelite Co.</td><td>MS-9096UZ</td><td></td></tr><tr><td>Regenova Bio 3D Printer</td><td>Cyfuse Biomedical K.K.</td><td>N/A</td><td>www.cyfusebio.com/en/</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Troponin T Antibody</td><td>Thermo Fisher</td><td>701620</td><td></td></tr><tr><td>Connexin 43 (Cx43) Antibody</td><td>Chemicon</td><td>MAB3068</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Thermo Fisher</td><td>P36935</td><td></td></tr></tbody></table>

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.

At the end of step 4.4 (co-culture), the cells in each well should aggregate at the bottom of the ultra-low attachment 96-well U-bottom plates to form spheroids by gravity. These spheroids contain hiPSC-CM, HCFs, and HUVECs, and can be visually inspected under light microscopy, where they should appear circular by two-dimensional projection (Figure 1). At the end of step 6.3, the 3D bioprinted cardiac patch should contain tissue voids, due to needle holes created by the needle array (Figure 2, left). At this point, the boundaries between the spheroids should have become indistinct and the patch should have begun to beat spontaneously, exhibiting mechanical integration of spheroids. At the end of step 6.5, the tissue voids should be filled in by surrounding tissue (Figure 2, right), while the 3D bioprinted cardiac patch should continue to beat spontaneously.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55438/55438fig1.jpg" /> 
Figure 1: Creation of mixed cell multicellular spheroids. hiPSC-CM, HCFs, and HUVECs were mixed at a hiPSC-CM:HCF:HUVEC cell ratio of 70:15:15, and the cell mixture was distributed into ultra-low attachment 96-well U-bottom plates. Within 24 h, mixed cell multicellular spheroids (left, right) formed, one in each well. Scale bar = 500 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55438/55438fig1large.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/55438/55438fig2.jpg" /> 
Figure 2: Creation of cardiac tissue exhibiting mechanical integration of spheroids. A 3D bioprinted cardiac patch immediately after removal from the needle array with visible needle holes (left). At this time, the boundaries between the spheroids had already become indistinct and the patch had already begun beating spontaneously, thus the spheroids had become mechanically integrated. Three days after removal from the needle array, the tissue voids caused by the needle array were filled in by surrounding tissue (right), and the patch continued beating spontaneously. Scale bar = 1 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55438/55438fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

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. 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 ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.9K Views.  Johns Hopkins Hospital. 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.

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

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. ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

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. ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

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 ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

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 ...

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

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Chapters in this video

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

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

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

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

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

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

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