This research was supported by the Houston Endowment grant and the Texas Emerging Technology Fund. The authors acknowledge the organ procurement agency LifeGift, Inc. and the donor&#39;s families for making this study possible.

All experiments adhered to the ethics committee guidelines from the Texas Heart Institute.
1. Organ Preparation
NOTE: In collaboration with LifeGift, a nonprofit organ procurement organization in Texas (http://www.lifegift.org), donated human hearts not suitable for transplant were used for research with approved consent.
<ol>
	<li>To procure hearts, intravenously infuse 30,000 U heparin to the hearts. Securely suture cardioplegia cannula in the aorta and attach a clamped perfusion line. Perforate the inferior vena cava (IVC) to vent the right heart. Cut either the left superior pulmonary vein or the left atrial appendage to vent the left chambers of the heart.</li>
	<li>Infuse 1 L of cardioplegia or heparinized saline. Dissect aortic arch branches, superior vena cava (SVC) and other pulmonary veins to release the heart from any vascular or surrounding tissue attachment. Submerge the well-heparinized heart in iced saline solution.</li>
	<li>Inspect the donated human heart (both anteriorly and posteriorly, Figure 1). Place the heart on a dissecting tray and inspect for any structural damage or anatomical malformations. If liver and/or lungs were procured for transplantation, the heart may present with a short inferior vena cava and/or absence of left atrial posterior wall.</li>
	<li>Perform internal inspection for possible defects - atrial septal defect (ASD), ventricular septal defect (VSD), or valve (aortic, pulmonary, mitral, tricuspid) malformation.</li>
	<li>If a septal defect is present, correct it with appropriate sutures (Figure 2A, 2B). The correction of septal defects is needed to monitor decellularization progress via pulmonary artery (PA) outflow turbidity measurement. Correction of the septal defect removes left to right shunt, hence, the outflow from PA represents the outflow from the coronary circulation through the coronary sinus.</li>
	<li>Ligate superior and inferior vena cava with 2-0 silk suture (Figure 2C).</li>
	<li>Dissect the aorta (Ao) away from the main PA (Figure 2D) for subsequent cannulation.</li>
	<li>Insert connectors, based on the diameter of the vessel, (Figure 3) into Ao and PA and secure them with 2-0 silk sutures (Figure 4A).</li>
	<li>Insert a tubing line through the left atrium (Figure 4B) and toward the left ventricle (LV) (Figure 3), using one of the pulmonary vein orifices.</li>
	<li>Connect an infusion line to the connector placed in the Ao and the outflow line to the one in the PA (Figure 3).</li>
	<li>Place the prepared heart into a polyester pouch in inverted orientation (upside down).</li>
	<li>Place the pouch with heart into a perfusion container and close the lid (Figure 4C).</li>
	<li>Connect each of the lines to the respective ports in the rubber stopper (based on the diameter of container) and insert it to the lid of the perfusion container to seal the polyester pouch (Figure 4C and Figure 5B).</li>
	<li>Perfuse 1x phosphate buffered saline (PBS) (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4 in distilled water, pH 7.4) via the infusion port of the rubber stopper to verify outflow from the PA and from the line inserted into LV.</li>
	<li>Use this flow to clean the organ of any residual traces of blood in the vasculature. If flow is not observed, tighten the connection lines as they might be loose.</li>
</ol>
2. System Setup and Organ Decellularization Procedure
<ol>
	<li>Assemble the bioreactor and place in an upright orientation (Figure 5). The perfusion system includes a personal computer (PC), proportional-integral-derivative (PID) controller, a peristaltic pump for Ao infusion, a perfusion bioreactor, a pressure head container for LV perfusate retention (2L aspirator bottle with bottom sidearm), and a peristaltic pump to drain excess fluid from the pressure head container and to collect the outflow from PA.</li>
	<li>In the rubber septum, connect the infusion line, pressure-head line, PA-outflow line and bioreactor draining line to the rubber cap surface ports placed on top of the perfusion bioreactor (Figure 5A).</li>
	<li>Decellularize hearts under constant pressure of 120 mmHg measured at the aortic root. Mean pressure of the LV should be within 14-18 mmHg throughout the whole decellularization process.</li>
	<li>Decellularize hearts as follows: 4 h of hypertonic solution (500 mM NaCl), 2 h of hypotonic solution (20 mM NaCl), 120 h of sodium dodecyl sulfate (1% SDS) solution, and a final wash with 120 L of 1X PBS (Figure 6A).</li>
	<li>Decellularize the hearts under constant pressure control (120 mmHg). Infusion flow rate into Ao is heart-dependent and is, on average, 98.06&#177;16.22 mL/min for hypertonic solution, 76.14&#177;7.90 mL/min for hypotonic solution, 151.50&#177;5.76 mL/min for SDS, and 185.24&#177;7.10 mL/min for PBS. The total consumed volume of each reagent averages 23.36&#177;5.70 L for hypertonic solution and 9.13&#177;1.26 L for hypotonic solution.</li>
	<li>Recirculate the final 60 L of 1% SDS (1 L per gram of heart weight) until the end of SDS perfusion. Figure 6A shows a timeline for the decellularization process, eliciting endpoints for data collection: flow rate monitoring (Ao and PA) and outflow collection (PA and non-PA) from pressure head during decellularization.</li>
	<li>Since the SVC and IVC are ligated, it is reasonable to assume that all fluid collected from the PA is the result of actual coronary perfusion (Figure 5B). Determine coronary perfusion efficiency by directly dividing the flow rate of perfusate out of the PA by the flow rate of infused solution into Ao: 
	Coronary perfusion efficiency = <img alt="Equation" src="/files/ftp_upload/58123/58123eq1.jpg" /> (%).</li>
	<li>Perform comparative analysis of the perfusate obtained from the PA and the LV and the infused solutions in duplicate by loading 200 &#956;L per well in a clear bottom 96-well plate and reading absorbance at 280 nm. The absorbance value, selected empirically after trying different values, was found to give the best normalized values.</li>
	<li>Use the turbidity of clean infused reagent as the control. The turbidity of the outflow perfusate represents washout of cell debris and can be quantified instantly during decellularization as a tracking tool of the process.</li>
	<li>During the final wash with 10 L of 1X PBS, add 500 mL of sterile neutralized 2.1% peracetic acid solution, neutralized with 10N NaOH, leading to a 0.1% peracetic acid solution (v/v) in PBS. Use this solution to sterilize the scaffold.</li>
</ol>
3. Evaluation of Decellularized Hearts
NOTE: After decellularization, representative hearts will be used for coronary angiogram imaging and biochemical assays.
<ol>
	<li>Perform coronary angiography of the representative decellularized human heart to examine the intactness of the coronary vasculature. Briefly, using a fluoroscope, image decellularized human heart after injection of contrast agent through coronary ostial cannula in the main right and left coronary arteries.</li>
	<li>Dissect the decellularized heart to get samples from 19 areas to evaluate the remaining deoxyribonucleic acid (DNA), glycosaminoglycan (GAG) and SDS levels in decellularized tissues. Remove the base of the heart from ventricles and dissect the ventricles into 4 equal sections (Figure 6C). Divide each section into the anterior and posterior right ventricle (RV), the anterior and posterior LV, and the interventricular septum (IVS). The tissue containing the apex is dissected into the LV and RV for sampling.</li>
	<li>Cut tissue samples for DNA, GAG and SDS assays (~15 mg of wet weight).</li>
	<li>Extract double-stranded DNA (dsDNA) by digesting samples in 1 M NaOH for 3 h at 65 &#176;C and adjust pH to 7 using 10x Tris-EDTA (TE) buffer and 1 M hydrochloric acid (HCl).</li>
	<li>Quantify dsDNA using a dsDNA Assay kit with a calf thymus standard (see Table of Materials). Read samples in duplicate using a fluorescence microplate reader (excitation at 480 nm and emission at 520 nm). Calculate the percentage of residual dsDNA in the decellularized hearts by comparing dsDNA concentration in each tissue to that in cadaveric (% cadaveric).</li>
	<li>Obtain sulfated GAGs into solution by digesting tissue samples in a papain extraction solution (0.2 M sodium phosphate buffer with EDTA disodium salt, cysteine HCl, sodium acetate, and papain) at 65 &#176;C for 3 hours. Measure GAG content (in duplicate) by using a Glycosaminoglycan Assay Kit.</li>
	<li>Lyophilize samples for SDS assay in a heated vacuum and measure dry weight. Add 200 &#181;L of ultrapure water to each dried sample and homogenize to extract the residual SDS into solution. Mix this SDS solution to chloroform and a methylene blue solution (12 mg of methylene blue in 1 L of 0.01 M HCl). The SDS will separate in the organic layer by binding to the methylene blue dye.</li>
	<li>Using a fluorescence microplate reader, read the absorbance (655 nm) of the standards and samples in duplicate to calculate the residual SDS. Normalize this SDS value to tissue dry weight.</li>
	<li>Image samples from thick regions (i.e., LV, RV and septum) of human heart with nonlinear optical microscopy (NLOM) to confirm cellular removal after decellularization. The NLOM setup is detailed in our previous publications18,19,20. NLOM enables us to image cell, elastin, collagen and myosin fibers through its two-photon fluorescence (TPF) and second harmonic generation (SHG) channels without using any exogenous stain or dye21.</li>
</ol>

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Dr. Taylor is the founder and shareholder in Miromatrix Medical, Inc. This relationship is managed in accordance with the conflict of interest policies by the University of Minnesota and Texas Heart Institute; the other authors have no conflict of interest to disclose.

To our knowledge, this is the first study to report inverted decellularization of human hearts inside a pressurized pouch with time-lapse monitoring of flow rate and cell debris removal. The pericardium-like pouch keeps the orientation of the heart stable throughout the decellularization procedure. Submerging and inverting the whole hearts inside a pouch prevents dehydration and minimizes excessive strain on the aorta (from heart weight) when compared to the conventional upright Langendorff perfusion decellularization me...

Heart failure leads to high mortality in patients. The ultimate treatment option for end-stage heart failure is allo-transplantation. However, there is a long wait-list for transplantation due to the shortage of donor organs, and patients face post-transplantation hurdles that range from life-long immunosuppression to chronic organ rejection1,2. Bioengineering functional hearts by repopulating decellularized human-sized hearts with a patient&#39;s own cells could circumvent these hurdles3.
A major step in &#34;engineering&#34; a heart is the creation of a scaffold with appropriate vascular and parenchymal structure, composition and function to guide the alignment and organization of delivered cells. In the presence of the appropriate framework, cells seeded on the scaffold should recognize the environment and perform the expected function as part of that organ. In our opinion, decellularized organ extracellular matrix (dECM) comprises the necessary characteristics of the ideal scaffold.
By utilizing intrinsic vasculature, complex whole-organ decellularization can be achieved via antegrade or retrograde perfusion4 to remove cellular components while preserving the delicate 3D extracellular matrix and vasculature2,5,6,7. A functional vasculature is important in bioengineering whole organs just as it is in vivo, for nutrient distribution and waste removal8. Coronary perfusion decellularization has been proven to be effective in creating decellularized hearts from rats4, or pigs4,7,9,10,11,12,13, and humans5,7,14,15,16. Yet, integrity of the valves, atria and other &#34;thin&#34; regions can suffer.
Human-size decellularized heart scaffolds can be obtained from pigs using pressure control7,9,10,11,12 or infusion flow rate control13,17 and from human donors using pressure control5,7,14,15. Decellularization of human donor hearts occurs over 4-8 days under pressure controlled at 80-100 mmHg in upright orientation5,15,16 or over 16 days under pressure controlled at 60 mmHg14. Under antegrade, pressure-controlled decellularization, the aortic valve competency plays a crucial role in maintaining coronary perfusion efficiency and stable pressure at the aortic root. Our previous work revealed that the orientation of the heart influences its coronary perfusion efficiency during the decellularization procedure and therefore the scaffold integrity in the end9.
As a continuation of our previous work9, we introduce a novel concept wherein a pericardium-like pouch is added to improve whole-heart decellularization. We describe the decellularization of human hearts placed inside pressurized pouches, inversely oriented, and under pressure controlled at 120 mmHg at the aortic root. This protocol includes monitoring the flow profile and collection of outflow media throughout the decellularization procedure to evaluate coronary perfusion efficiency and cell debris removal. Biochemical assays are then performed to test the effectiveness of the method.

<table>
	<tbody>
		<tr>
			<td>2-0 silk suture</td>
			<td>Ethicon</td>
			<td>SA85H</td>
			<td>Suture used to ligate superior and inferior vena cava</td>
		</tr>
		<tr>
			<td>1/4&#34; x 3/8&#34; connector with luer</td>
			<td>NovoSci</td>
			<td>332023-000</td>
			<td>Connect aorta and pulmonary artery</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing</td>
			<td>Cole-Parmer</td>
			<td>HV-96410-16</td>
			<td>Tubing to connect heart chambers/veins</td>
		</tr>
		<tr>
			<td>infusion and outflow line</td>
			<td>Smiths Medical</td>
			<td>MX452FL</td>
			<td>For flowing solutions through the vasculature</td>
		</tr>
		<tr>
			<td>Polyester pouch (Ampak 400 Series SealPAK Pouches)</td>
			<td>Fisher scientific</td>
			<td>01-812-17</td>
			<td>Pericardium-like pouch for containing heart during decellularization</td>
		</tr>
		<tr>
			<td>Snapware Square-Grip Canister</td>
			<td>Snapware</td>
			<td>1022</td>
			<td>1-liter Container used for perfusing heart</td>
		</tr>
		<tr>
			<td>Black rubber stoppers</td>
			<td>VWR</td>
			<td>59586-162</td>
			<td>To seal the perfusion container</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Harvard Apparatus</td>
			<td>881003</td>
			<td>To pump fluid through the inflow lines and to drain fluids</td>
		</tr>
		<tr>
			<td>2 L aspirator bottle with bottom sidearm</td>
			<td>VWR</td>
			<td>89001-532</td>
			<td>For holding solutions/perfusate</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay kit</td>
			<td>Life Technologies</td>
			<td>P7589</td>
			<td>For quantifying dsDNA</td>
		</tr>
		<tr>
			<td>Calf thymus standard</td>
			<td>Sigma</td>
			<td>D4522</td>
			<td>DNA standard</td>
		</tr>
		<tr>
			<td>Blyscan Glycosaminoglycan Assay Kit</td>
			<td>Biocolor Ltd</td>
			<td>Blyscan #B1000</td>
			<td>GAG assay kit</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200 Pro</td>
			<td>For analytical assays</td>
		</tr>
		<tr>
			<td>GE fluoroscopy</td>
			<td>General Electric</td>
			<td>OEC 9900 Elite</td>
			<td>Angiogram</td>
		</tr>
		<tr>
			<td>Visipaque</td>
			<td>GE</td>
			<td>13233575</td>
			<td>Contrast agent</td>
		</tr>
	</tbody>
</table>

decellularization of whole human heart inside a pressurized pouch in an inverted orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

After a 7-day decellularization with antegrade aortic perfusion under constant pressure of 120 mmHg, the human heart turned translucent (Figure 6B). The heart was grossly dissected into 19 sections for biochemical (DNA, GAG and SDS) analysis (Figure 6C) to evaluate the final decellularized product.
Throughout the decellularization process, infusion flow rate of differen...

Watch this Scientific Journal Video about Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation at JoVE.com

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

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Research

JoVE Journal

Bioengineering

11.2K Views.  Texas Heart Institute. This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Video: Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

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

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...