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. 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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 border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">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>

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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 ...

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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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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. ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

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 ...

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 ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...