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 border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">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/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/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/files/ftp_upload/58252/58252fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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. ...

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...