The authors would like to acknowledge Brogan Guest, Michelle Weaver, and Kristen Lippert. Funding for this study was provided by NIH Grant R03EB009237, as well as NIH Training Grants T32EB001026-06 from the National Institute of Biomedical Imaging And Bioengineering and T32HL076124-05.

1. Tissue Preparation and Experiment Setup
<ol>
	<li>Harvest porcine organ immediately after euthanasia from an abattoir or research facility and rinse off excess blood. Trim the heart of excess fat and tissue, keeping the atria and aorta intact. Trim away fat to separate the pulmonary artery from the aorta. If there are any cuts in the tissue, discard appropriately.</li>
	<li>Wrap each heart individually in freezer paper and store all tissue in a -80 &deg;C freezer for at least 24 hr to ensure complete freezing.</li>
...

<ol>
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The current study described methodology for consistent and efficient decellularization of a porcine heart. The protocol was a modification to a previously published report 1, and included longer exposure to flow and increased pressure, which provided more repeatable results. The resulting decellularized tissue met all of the published criteria for successful decellularization of tissue 2. Frequent solution changes were performed to limit the reintroduction of cellular material to the tissue,...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>15090</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-500</td>
 <td></td>
 </tr>
 <tr>
 <td>NaN3</td>
 <td>Sigma</td>
 <td>S2002-500G</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma</td>
 <td>X100-1L</td>
 <td></td>
 </tr>
 <tr>
 <td>10X PBS</td>
 <td>Fisher</td>
 <td>BP399-20</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Deoxycholate</td>
 <td>Sigma</td>
 <td>D6750-500G</td>
 <td></td>
 </tr>
 <tr>
 <td>Peracetic Acid</td>
 <td>Pfaltz and Bauer</td>
 <td>P05020</td>
 <td>35% CAS# 79-21-0</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Pharmco</td>
 <td>111000200</td>
 <td></td>
 </tr>
 <tr>
 <td>Masterflex Pump Drive</td>
 <td>Cole Parmer</td>
 <td>SI-07524-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Masterflex Tubing</td>
 <td>Cole Parmer</td>
 <td>96400-18</td>
 <td>Size 18</td>
 </tr>
 <tr>
 <td>Barbed Reducer</td>
 <td>Cole Parmer</td>
 <td>EW-30612-20</td>
 <td></td>
 </tr>
 <tr>
 <td>4L Beaker</td>
 <td>Fisher Scientific</td>
 <td>02-540T</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

The effect of decellularization on whole porcine hearts naturally varies due to differences in size, pressures, and vessel arrangement. Therefore, the exact composition of the derived extracellular matrix scaffolds will not be the same from heart to heart. The completion of the described protocol will yield a heart that appears white or translucent, indicating the loss of cellular material. However, it is widely accepted that a tissue can be considered "decellularized" based on the combination of a few more quantit...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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 ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.7K visualizações.  McGowan Institute for Regenerative Medicine. Um método para rápida e completamente remover componentes celulares a partir de um coração porcino intacta através da perfusão retrógrada é descrito. Este método produz um sítio específico cardíaco matriz extracelular andaime que tem o potencial para uso em várias aplicações clínicas.