Name: decellularization of whole human heart inside a pressurized pouch in an inverted orientation
Uploaded: 2018-11-26T13:30:00
Description: Watch this Scientific Journal Video about Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation at JoVE.com

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...

<ol>
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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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