The authors would like to thank Marlijn Jansen, Joyce Visser, and Martijn van Nieuwburg for their assistance with the animal experiments. We greatly acknowledge Martijn Froeling and Anke Wassink for their assistance with the MRI imaging.

The in vivo experiment was conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Research. The experiment was approved by the local Animal Experiments Committee.
1. Preparation of Injectable and Embedding Solution
<ol>
	<li>Prepare the injectable gel.
	<ol>
		<li>Prepare 1 mL of ureido-pyrimidinone (UPy) gel in accordance to previously-described protocols12,13.</li>
		<li>Add superparamagnetic iron oxide particles (SPIOs) to the solution to get a concentration of 15 &#181;g/mL and stir the mixture for 5 min for uniform distribution.</li>
		<li>Add the fluorescent microbeads to the solution to get a concentration of 10,000 beads/mL and stir the mixture for 5 min for uniform distribution.</li>
		<li>Store the resulting mixture at room temperature in a dark environment. Warm and vortex or stir the solution shortly before the injection procedure.</li>
	</ol>
	</li>
	<li>Prepare the embedding solution.
	<ol>
		<li>Start with tap water at room temperature and add agarose (agar) to a concentration of 4 wt%.</li>
		<li>Slowly heat up the solution to the boiling point using a microwave oven and stir frequently during heating. Upon reaching the boiling point, store and keep the agar solution above 70 &#176;C for 2 h to allow trapped air to surface.</li>
		<li>Allow the agar to cool down at room temperature to a temperature between 50 and 60 &#176;C until the time of embedding.</li>
	</ol>
	</li>
</ol>
2. Injection Procedure
<ol>
	<li>Perform premedication (anti-arrhythmic agents, anti-platelet therapy, and pain medication), anesthesia, venous access, and intubation, as previously described14.</li>
	<li>Perform injections using an intramyocardial injection catheter (Table of Materials). For each injection, 0.2 mL of the mixture is injected in one bolus at a constant rate of approximately 0.3 mL/min using an injection device. Place the injections at different positions along the IBZ12.</li>
	<li>Administer 0.2 mL/kg (1.0 mmol/mL) of a gadolinium-based contrast agent 15 min prior to euthanizing the animal.</li>
	<li>Administer 20 mL of 7.5% potassium chloride intravenously to euthanize the animal.</li>
	<li>Secure mediastinal access following protocol steps 8.2 - 8.3, as described by Koudstaal et al.14. Cut the inferior caval vein 5 cm from the right atrium and remove outflowing blood with a suction device. Excise the heart and rinse it with 0.9% saline at room temperature.</li>
</ol>
3. Embedding Procedure
<ol>
	<li>Prepare the heart.
	<ol>
		<li>Remove the pericardium from the heart while keeping the atria and ventricles intact. Dissect the ascending aorta &#177; 1 cm above the aortic valve using Klinkenberg scissors. Cut the inferior caval vein &#177; 1 cm from the atrium, and do the same for the pulmonary veins.</li>
		<li>Suture the apex of the heart to the bottom of a plastic embedding container (17 x 15 x 15 cm, W x D x H) using a 2-0 suture to prevent floatation of the heart during embedding (Figure 1A).</li>
		<li>Suture the remaining part of the aorta to the rims of the container using 2-0, making sure that the heart is centered and not touching the walls of the container (Figure 1B).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54974/54974fig1.jpg" /> 
Figure 1: Schematic Overview and Photograph of the Embedding Container. (A) Schematic overview of the embedding process. The heart (red) is secured in the container (blue) using sutures. After filling the heart with the agar solution, the space around the heart is filled. Finally, two rigid plastic tubes (yellow) are positioned in the container, next to but not touching the heart, to serve as a reference during image registration. (B) Photograph of a heart secured in the embedding container. The sutures are clamped to the rim of the container using mosquito clamps. <a href="http://cloudfront.jove.com/files/ftp_upload/54974/54974fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Embed the heart in an end-diastolic-like geometry. 
	NOTE: The prevention of air bubble creation is necessary. If large air bubbles present in the agar solution, keep the agar at 40 &#176;C, allowing the air bubbles to surface.
	<ol>
		<li>Clamp the inferior caval vein using mosquito clamps. Slowly inject the liquid agar using a 50-mL syringe in the right atrium via the superior caval vein until both the right atrium and ventricle are completely filled.</li>
		<li>Clamp the pulmonary veins using mosquito clamps. Gently pass an agar-filled 50-mL syringe retrogradely through the aortic valves. Slowly inject the solution in the left ventricle (LV) until the LV and the left atrium are completely filled. After filling the LV, clamp the aorta to keep the agar in the LV.</li>
		<li>Pour the remaining agar into the container until the heart is fully covered. Place two rigid plastic tubes within the embedding container to serve as reference structures for later image registration (Figure 1A). Make sure the tubes do not touch the walls of the container or the heart.</li>
		<li>Let the agar solidify at 2 - 7 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Image Acquisition
<ol>
	<li>Perform transversal ex vivo MRI scans of the heart that is embedded in the container.
	<ol>
		<li>Place the container with the embedded heart inside a head coil (Table of Materials).</li>
		<li>Angulate the slices parallel to the bottom of the container. Use the same orientation and angulation in each ex vivo MRI sequence.</li>
		<li>To visualize myocardium, perform a fluid-attenuated inverse recovery (FLAIR) sequence with the following parameters: repetition time [TR]/echo time [TE] = 10 s/140 ms, flip angle = 90&#176;, pixel size = 0.5 x 0.5 mm, field of view [FOV] = 169 x 169 mm, 320 x 320 matrix, and 3-mm slice thickness.</li>
		<li>To visualize the myocardial infarction, perform a late-gadolinium enhanced (LGE) sequence with the following parameters: [TR]/[TE] = 5.53 ms/1.69 ms, flip angle = 25&#176;, pixel size = 1.0 x 1.0 mm, [FOV] = 169 x 169 mm, 176 x 176 matrix, and 3-mm slice thickness.</li>
		<li>To visualize SPIOs, perform a T2*-weighted gradient echo sequence with the following parameters: [TR]/[TE] = 88.7 ms/15 equally-distributed TEs with a range of 1.9 - 24.6 ms, flip angle = 15&#176;, pixel size = 0.5 x 0.5 mm, [FOV] = 169 x 169 mm, 320 x 320 matrix, and 3-mm slice thickness.</li>
	</ol>
	</li>
	<li>Tissue processing
	<ol>
		<li>Turn the container upside down and allow air between the agar and the sides of the container to remove the solid agar solution, including the heart, from the container. Remove the plastic rods from the solid agar.</li>
		<li>Section the agar block containing the heart in 5-mm slices from the apex to the base of the heart using a meat slicer. Keep the angulation of the cut slices the same as in the acquired MR images by cutting parallel to the bottom of the agar block.</li>
		<li>Stain the agar slices (including the heart) for 15 min in 1 wt% of 2,3,5-triphenyltetrazoliumchloride (TTC) dissolved in 0.9% saline at 37 &#176;C, and photograph the slices on both sides from a perpendicular view (Figure 2A). Next, carefully rinse the slices in 0.9% saline. 
		NOTE: In this study, we used a dSLR setup with an appropriate lens/objective, a tripod, and uniform lighting. However, the photographs served only as a control for the assessment of the scar region, so we could have used a different setup.</li>
	</ol>
	</li>
	<li>Fluorescence imaging 
	NOTE: Depending on the excitation and emission wavelengths of the fluorescent microbeads, select the appropriate filter block and excitation lasers (e.g., the red microbeads used here have excitation and emission wavelengths of 580 nm and 605 nm, respectively; therefore, the selected excitation laser and bandpass filters were set to 532 nm, 580/30 nm and 610/30 nm, respectively).
	<ol>
		<li>Select fluorescence-mode imaging on the variable-mode scanner. Set the photomultiplier tube to 430 V or equivalent and the pixel size to 100 x 100 &#181;m. Select an excitation laser (532 nm) closest to the excitation wavelength of the fluorescent microbeads.</li>
		<li>For the first filter block, select a bandpass filter (580/30 nm) that overlaps with the emission wavelength of the injected fluorescence beads (channel 1). Select a bandpass filter for the second filter block (610/30) outside the emission wavelength (channel 2). 
		NOTE: The second filter block serves as a negative control and to remove auto-fluorescence while keeping the injection sites intact.</li>
		<li>Scan both sides of the agar slices in the fluorescence mode of the variable-mode laser scanner using the two channels. Make sure that each slice is completely scanned, including the reference holes.</li>
	</ol>
	</li>
</ol>
5. Post-processing
NOTE: The first step in image post-processing is the manual segmentation of the myocardium using in-house developed scripts to trace the endo- and epicardial borders, as well as the injection sites. This is the same for both MRI and fluorescence scans.
<ol>
	<li>Segment the myocardium in the MRI scans.
	<ol>
		<li>Segment the endocardial and epicardial LV borders on the FLAIR MRI sequence images.</li>
		<li>Copy the LV segmentation from step 5.1.1 to the LGE-MRI dataset and segment the scar on the LGE MRI sequence.</li>
		<li>Copy the myocardium segmentation from step 5.1.1 to the T2*-weighted dataset and segment the SPIO depositions in the LV myocardium.</li>
	</ol>
	</li>
	<li>Process the fluorescence images and perform segmentations.
	<ol>
		<li>Load the files obtained from the variable-mode scanner and make a separate image of each cross-sectional heart slice.</li>
		<li>Flip the slices that were scanned in base to apex orientation and sort the fluorescence images into a stack for both channels that is oriented from apex to base.</li>
		<li>Segment the endocardial and epicardial LV borders on the fluorescence images.</li>
		<li>Segment the scar manually on the fluorescence images and use the LGE-MRI scan and the photographs to confirm scar morphology.</li>
		<li>Subtract the image stack of channel 2 from the image stack of channel 1 to exclude auto-fluorescence. Manually segment the fluorescent microbead depositions and use the T2* images for confirmation.</li>
	</ol>
	</li>
	<li>To create an anatomically-correct 3D geometry, perform a rigid registration of the slices in the image stack based on the reference structures (the holes created by the rigid tubes). Calculate and store the applied translation and rotation of each image.</li>
	<li>Apply the stored transformations to the image stacks and the segmentations. Linearly interpolate the segmentations of both sides of the slices to reconstruct the original slice thickness and to create a 3D model of the data.</li>
</ol>
6. Analysis
<ol>
	<li>Perform 2D and/or 3D measurements of the distance between the centers of the injection sites and the IBZ to assess the injection accuracy. Measure the distance along the endocardial border of the LV segmentation. In Figure 2C and 2F, an example of the 2D and 3D measurements is indicated by the red line.</li>
</ol>

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The authors have nothing to disclose.

Whole-heart 3D myocardial tissue processing according to this protocol provides a structured method that enables the 3D analysis of the infarct, the IBZ, and the performed injections with respect to the cardiac anatomy. The filling volume of the heart depends on the desired analysis. In this study, to assess the injection accuracy, we aimed to fill the heart to resemble the end-diastolic geometry as closely as possible. To enforce this, the LV apex is fixed to the bottom of the container and the LV is filled with agar wh...

Ischemic heart disease has been the world&#39;s leading cause of death for the past decades1. Acute treatment after myocardial infarction aims to restore blood flow to the myocardium via percutaneous coronary intervention or coronary artery bypass grafting. In severe infarctions, a large area of the myocardium is scarred, and these cases often result in ischemic heart failure (HF)2. Current treatment options for HF focus on prevention and the preservation of cardiac function for the HF patients, but not on regeneration.
In the last decade, cardiac regenerative therapies have been investigated as a treatment option for HF3. This therapy aims to deliver biologicals, such as stem cells or growth factors, directly to the injured myocardium to induce revascularization, cardiomyocyte protection, differentiation, and growth4. For optimal therapeutic effect, it is hypothesized that the biological must be injected in the infarct border zone (IBZ) to facilitate good tissue perfusion for the survival of the biological and for optimal effect to the target zone5,6. Multiple techniques have been developed to perform identification and visualization of the IBZ to guide intramyocardial injections7,8,9,10,11. Besides identification and visualization of the IBZ, the delivery also relies on the biomaterials and injection catheters used. To validate the injection accuracy of the delivery techniques, an accurate and reproducible quantification method is required.
We have developed a protocol for whole-heart myocardial tissue processing that offers two-dimensional (2D) and three-dimensional (3D) imaging, which can be used for qualitative and quantitative study aims. The protocol covers the embedding process and the digital image analysis. In this paper, we demonstrate a protocol for the assessment of the targeting accuracy of intramyocardial injections in the IBZ in a large porcine model of chronic myocardial infarction.

<table><tbody><tr><td>0.9% Saline</td><td>Braun</td><td></td><td></td></tr><tr><td>Agarose</td><td>Roche Diagnostics</td><td></td><td>Scientific grade multipurpose agar</td></tr><tr><td>Biomolecular fluorescence scanner Typhoon 9410&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Embedding container</td><td></td><td></td><td>Plastic, dimensions 17 x 14.5 x 14 cm</td></tr><tr><td>FluoSpheres Polystyrene Microspheres</td><td>Invitrogen</td><td>F8834</td><td>red, 10 &amp;micro;m</td></tr><tr><td>Gadolinium</td><td>Gadovist</td><td></td><td>1.0 mmol/mL</td></tr><tr><td>dS 32 channel head coil</td><td>Philips</td><td></td><td>Or similar</td></tr><tr><td>Matlab</td><td>Mathworks</td><td></td><td>To insure compatability 2015a or newer</td></tr><tr><td>Meat slicer</td><td>Berkel</td><td></td><td></td></tr><tr><td>Myostar injection catheter</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>Super paramagnetic iron oxide particles</td><td>Sinerem</td><td></td><td></td></tr><tr><td>Triphenyl-tetrazolium chloride</td><td>Merck</td><td></td><td></td></tr><tr><td>UPy-PEG10k</td><td></td><td></td><td></td></tr><tr><td>Vicryl 2-0</td><td>Ethicon</td><td></td><td></td></tr></tbody></table>

3d whole-heart myocardial tissue analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

Tissue Embedding
Through the embedding process, an end-diastolic-like geometry was established. The agar successfully adhered to the heart tissue, enabling the tissue to be sliced at the desired angulation with equal slice thicknesses (Figure 2A and 2C).
Scar- and Injection-site Assessment
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Watch this Scientific Journal Video about 3D Whole-heart Myocardial Tissue Analysis at JoVE.com

3D Whole-heart Myocardial Tissue Analysis

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

3d-whole-heart-myocardial-tissue-analysis

Research

JoVE Journal

Medicine

8.7K Views.  University Medical Center Utrecht. This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction. 

Video: 3D Whole-heart Myocardial Tissue Analysis

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioengineering

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Developmental Biology

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

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Biology

Mixed Reality Technology and Three-Dimensional Printing in Teaching: Heart Anatomy as an Example

Mixed reality technology and three-dimensional (3D) printing are becoming more and morecommon in the field of medicine. During the COVID-19 pandemic and immediately after the restrictions had beeneased, many innovations were implemented in the teaching of future doctors. There was also interest in immersive techniques and 3D printing technology in anatomy teaching. However, these are not common implementations. In 2023, 3D prints and holograms in mixed reality technology were prepared for classes focused on the structure of the heart. They were used to teach students, who, with the support of engineers, could learn about the detailed structure of the heart and familiarize themselves with the new technologies that support the traditional model of learning on human cadavers. Students findthis possibility to be highly valuable. The article presents the process of preparing materials for classes and further implementation possibilities. The authors see an opportunity for the development of the presented technologies in students'teaching at various levels of education and the justification for increasingly widespread implementation.

Here, we describe a protocol for constructing a heart model from scratch based on computed tomography and present it to medical students using three-dimensional (3D) printing and mixed reality technology to learn anatomy.

Mixed reality technology and three-dimensional (3D) printing are becoming more and morecommon in the field of medicine. During the COVID-19 pandemic and ...

Comprehensive 3D Analysis of Human Hearts from Micro to Macro Scales

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results&#160;to illustrate progress in the research of human cardiac anatomy.

Author Spotlight: Advancing Human Cardiac Anatomy Through Multi-Scale Analysis of Hearts

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...

3D Whole-heart Myocardial Tissue Analysis

Summary

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Chapters in this video

Ultrasonic Assessment of Myocardial Microstructure

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

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

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

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Mesoscopic Optical Imaging of Whole Mouse Heart

Mixed Reality Technology and Three-Dimensional Printing in Teaching: Heart Anatomy as an Example

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart