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>
	<li>When ready for use (usually less than 3 months), thaw one intact frozen porcine heart in Type 1 water overnight submerged in a 4 L beaker at 4 &deg;C.</li>
	<li>After the heart is completely thawed, pat the heart dry, weigh the heart, and record the weight. The heart of a market weight pig should weigh approximately 375-450 g.</li>
	<li>Connect size 18 Masterflex tubing to the &frac14;" end of a barbed reducer. Insert the barbed reducer and tubing inside the aorta. Place 2 hose clamps or secure zip ties around the aorta, just below the brachiocephalic trunk. The reducer and tubing must remain above the aortic valve, so the coronary arteries can be perfused (Figure 1).</li>
	<li>Use a 30 or 60 ml syringe to fill the tubing with Type I water. Insert the tubing within the cartridge of a Masterflex roller pump at its approximate midpoint. Submerge the inflow end of the tubing in the bottom of a 4 L beaker filled with 2.5 L of water and secure the tubing.</li>
	<li>Place the heart in the beaker filled with water, and prime the pump to remove air bubbles. If bubbles are observed coming from the aorta where the tubing is inserted, the aorta may need to be repositioned or secured with additional ties. An airtight seal is important to maintain adequate pressure during the decellularization process (Figure 2).</li>
	<li>Place the 4 L beaker containing 3 L of a 0.2% Trypsin/0.05% EDTA/0.05% NaN3 solution on stir plate and warm it to 37 &deg;C in preparation of the decellularization process.</li>
</ol>
2. Tissue Rinses
<ol>
	<li>Set the pump to a flow rate of 400 ml/min, ensuring that the correct tubing size is selected. Flush the heart with Type I water for 15-25 min. As the pump is started, the heart should swell and effuse blood from the ventricles. Fresh solution should be substituted every 5-10 min, or as needed based on the amount of blood removed from the heart. If blood is not effused from the heart, adjust the tubing and clamps as necessary.</li>
	<li>Stop the pump and transfer the heart to a separate beaker filled with 2X Phosphate Buffered Saline (PBS). After the tubing is submerged in solution, start the pump and increase the flow rate to 700 ml/min. The heart should remain in solution for 15 min, changing the solution every 5 min. Each solution change requires the pump to be stopped temporarily while the tissue and tubing is moved to the new beaker.</li>
	<li>Transfer the heart to Type I water for 10 min and increase the flow rate to 750 ml/min.</li>
</ol>
3. Decellularization and Solution Perfusion
<ol>
	<li>Transfer the heart to the beaker containing 0.2% Trypsin/0.05% EDTA/0.05% NaN3 at 37 &deg;C. Increase the pump speed to 1,200 ml/min and start the pump. Use a stir bar placed at the bottom of the beaker to circulate solution in the beaker. The heart should remain in the 0.2% Trypsin/0.05% EDTA/0.05% NaN3 solution at 37 &deg;C for a total of three hours. After 1 hr, increase the pump speed to 1,500 ml/min. After an additional hour, increase the pump speed to 1,800 ml/min. The tissue is slowly subjected to increased perfusion speeds to condition the tissue and prevent rupture of the vessels. The heart will swell and nearly double in size during this step of the protocol. The tissue will lose its natural color, progressing from the atria to the apex throughout the protocol (Figure 3).</li>
	<li>After each solution perfusion, a two step rinse is performed to remove cellular debris, chemical residue, and aid cell lysis. Each rinse consists of a 10 min rinse in Type I water followed by a 10 min rinse with 2X PBS solution at room temperature. Each wash consists of removal of solution from the original beaker, adding rinse solutions, and circulating the perfusate within the beaker containing the submerged heart. After the 0.2% Trypsin/0.05% EDTA/0.05% NaN3 solution, perfuse water at 1,900 ml/min and then perfuse 2X PBS at 1950 ml/min.</li>
	<li>Transfer the heart to a solution of 3% Triton X-100/0.05% EDTA/0.05% NaN3 at room temperature. Increase the pump speed to 2,000 ml/min and perfuse solution for one hour. Remove the solution from the beaker and replace with fresh solution, increase the pump speed to 2100 ml/min, and perfuse the fresh solution for an additional hour and a half, bringing the total time in 3% Triton X-100/0.05% EDTA/0.05% NaN3 to 2.5 hr.</li>
	<li>Rinse the tissue in Type I water at 2150 ml/min and 2X PBS at 2180 ml/min for 10 min each.</li>
	<li>Transfer the heart to a 4% Sodium Deoxycholate solution at room temperature. Increase the pump speed to 2,200 ml/min and perfuse solution for 3 hr.</li>
	<li>Rinse the tissue in Type I water at and 2X PBS at 2,200 ml/min for 15 min each, changing the solutions after 5-10 min for each solution. The described perfusion steps may be split over multiple days by performing the rinse step twice and storing the heart with attached tubing overnight at 4 &deg;C and submerged in Type I water.</li>
	<li>The following day, perform a 5 min rinse with Type I water at 750 ml/min, followed by a 5 min rinse in 1X PBS at 1,500 ml/min. The protocol may then be continued at the described flow rate in the proper solution.</li>
</ol>
4. Disinfection and Final Processing
<ol>
	<li>Transfer the heart to a 0.1% peracetic acid/4% ethanol solution and perfuse solution for 1.5 hr at 2,200 ml/min.</li>
	<li>The final rinses for the tissue are all performed at 2,200 ml/min. Perfuse the tissue with 1X PBS for 15 min, followed by two 5 min washes in Type I water. This series of rinses is repeated once more in order to complete the solution perfusion procedure.</li>
	<li>Turn the pump off and remove the heart from solution to drain the heart. Cut the ties from the aorta, remove all tubing, and place the heart in an empty beaker to drain for 1 hr. Excess liquid will need to be drained periodically. Lay the heart on an absorbent pad to fully drain the heart (Figure 4).</li>
	<li>After most of the water is removed, record the weight of the cardiac extracellular matrix (C-ECM). The heart can be expected to lose approximately 20-25% of its initial weight during the decellularization process.</li>
	<li>Dissect the right and left ventricles, as well as the ventricular septum for DNA quantification and histological processing in order to confirm complete decellularization of the tissue (Figure 5).</li>
	<li>Freeze the C-ECM at -80 &deg;C for at least 2 hr before lyophilization.</li>
</ol>

<ol>
	<li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. , (2008).</li><li>Petersen, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329, 538-541 (2010).</li><li>Uygun, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. , (2010).</li><li>Wainwright, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cardiac+extracellular+matrix+from+an+intact+porcine+heart.">Preparation of cardiac extracellular matrix from an intact porcine heart.</a> Tissue Eng. Part C Methods. 16, 525-532 (2010).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. , (2012).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Akhyari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quest+for+an+optimized+protocol+for+whole-heart+decellularization:+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities.">The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.</a> Tissue Eng. Part C Methods. 17, 915-926 (2011).</li><li>Weymann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+perfusion+decellularization+porcine+heart+model--generation+of+3-dimensional+myocardial+neoscaffolds.">Development and evaluation of a perfusion decellularization porcine heart model--generation of 3-dimensional myocardial neoscaffolds.</a> Circulation journal : official journal of the Japanese Circulation Society. 75, 852-860 (2011).</li><li>Cortiella, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (1089).</li><li>Remlinger, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrated+xenogeneic+decellularized+tracheal+matrix+as+a+scaffold+for+tracheal+reconstruction.">Hydrated xenogeneic decellularized tracheal matrix as a scaffold for tracheal reconstruction.</a> Biomaterials. 31, 3520-3526 (2010).</li><li>Sellaro, T. L., Ravindra, A. K., Stolz, D. B., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+hepatic+sinusoidal+endothelial+cell+phenotype+in+vitro+using+organ-specific+extracellular+matrix+scaffolds.">Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds.</a> Tissue Eng. 13, 2301-2310 (2007).</li><li>Wainwright, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+outflow+tract+repair+with+a+cardiac+biologic+scaffold.">Right ventricular outflow tract repair with a cardiac biologic scaffold.</a> Cells, tissues, organs. 195, 159-170 (2012).</li></ol>

Dr. Gilbert was on the Scientific Advisory Board at ACell, Inc. while the study was being done, and recently became the VP of Research and Development. ACell, Inc. sells urinary bladder matrix and has no commercial interest in the present study.

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, and the duration of exposure to each decellularization agent was minimized to reduce adverse effects on the ECM. During the beginning stages of the protocol, the perfusion rate was gradually increased to condition the tissue and allow for higher flow rates during the later stages of the protocol. Without conditioning the tissue in the early stages, the vasculature of the heart can rupture, making perfusion of the heart impossible. The protocol was used due to its efficiency, and no claims are made to its superiority over other protocols. The precise composition of decellularization agents and rates of perfusion may conceivably be varied to yield a protocol with better mechanical or biologic characteristics, but the general principles for delivery of the agents to the heart are applicable.
The preservation of the native three-dimensional structure of the heart was attributed to several procedures performed throughout the decellularization protocol. First, the tissue was trimmed and frozen upon arrival. Freezing promoted cell lysis and was important for pre-conditioning the tissue for the perfusion cycles. The tissue was thoroughly inspected for cuts and 2 cm intact intact aorta superior to the aortic valve. If any pericardium or epicardium was cut, the organ was discarded because the perfusate did not reach downstream regions of the heart, and the heart was not fully decellularized. Next, the tissue was fully thawed in type I water before use. The water allowed the tissue to relax as it thawed and also aided the removal of residual blood clots within the heart. Finally, as the tubing was inserted, care was taken to ensure that the aortic valve remained intact so that it formed a water-tight seal around the tubing, so that a proper pressure was maintained and that the solution entered the coronary arteries.
After each decellularization protocol was completed, a series of quality control measures were completed to ensure complete removal of cellular material. The current study verified that the protocol eliminated histologic staining for cell nuclei, showed that less than 50 ng of DNA was present per mg of dry weight of the tissue, and that any DNA was less than 200 bp in size 6. Previously published methods for cardiac decellularization showed similar levels of decellularization in DNA staining and quantification 2,5,9,10. Complete decellularization was accomplished in these studies using similar treatments of enzymes and detergents. However, in the present study, the length of exposure to each chemical was increased, there were more solution changes, and the flow rates were increased. The present protocol also increased the length of chemical rinses, potentially leading to more efficient removal of chemical residues from the extracellular matrix.
Recellularization of decellularized rat hearts with cardiac specific cells has yielded promising results in vitro 2,5. Whole organ perfusion decellularization allowed for maintenance of the native vasculature, which is critical in recellularization of the tissue. The inherent growth factors, matrix proteins, and three-dimensional fiber architecture also promoted proper cell attachment, migration, and signaling to reconstitute contractile myocardial tissue. Porcine cardiac extracellular matrix will be more difficult to recellularize due to the size of the scaffold and the number of cells necessary for proper cell communication and nutrient transport. However, patches cut from the cardiac matrix may be useful for in vivo applications. Multiple studies have shown the potential advantage of using a site-specific matrix for reconstruction of damaged tissue in animal models 11-13. Thus, an extracellular matrix scaffold derived from cardiac tissue is desirable for myocardial reconstruction applications. The inherent architecture of the cardiac tissue may present advantages over an ECM scaffold derived from another organ or an artificial biomaterial. A site-specific scaffold may support host cell infiltration and promote a constructive remodeling response, as opposed to scar tissue formation. To date, cardiac ECM patches have been investigated in vivo to reconstruct a defect created in the myocardial wall 14. Future studies will be performed in vitro to examine the ability of the scaffold to support cardiac cells seeded and cultured on the matrix. The methods described herein may also be applicable to decellularization of human hearts.
In conclusion, porcine heart decellularization is possible and the methods are straight-forward. Continued investigation of this material will provide insight into its potential for clinical use.

<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 quantitative parameters 8. A successful decellularization protocol will produce a matrix with less than 50 ng of double stranded DNA per mg of tissue (Figure 6). In order to avoid a host immune response upon implantation of the matrix, the remaining DNA should also contain less than 200 base pairs (Figure 7). To confirm these findings, Hematoxylin and Eosin staining should reveal the absence of nuclear staining in representative sections of the ventricles and ventricular septum (Figure 8). Masson's Trichrome further confirms the loss of cardiac muscle bundles and retention of collagen networks (Figure 9).
<img src="/files/ftp_upload/50059/50059fig1.jpg" alt="Figure 1"/> 
Figure 1. The barbed end of the tubing is inserted into the aorta of the native heart. The tubing must be secured with hose clamps or zip ties above the aortic valve to ensure perfusion through the coronary arteries.
<img src="/files/ftp_upload/50059/50059fig2.jpg" alt="Figure 2"/> 
Figure 2. The heart is submerged in water in a 4L beaker and air bubbles must be removed from the tubing. If bubbles are observed emerging from the aorta near the tubing, additional ties must be used to secure the tubing to the aorta in order to maintain adequate pressure in the tissue.
<img src="/files/ftp_upload/50059/50059fig3.jpg" alt="Figure 3"/> 
Figure 3. As solutions are perfused through the coronary arteries, the heart will lose its native color, progressing from the atria to the apex of the heart and localized around the coronaries.
<img src="/files/ftp_upload/50059/50059fig4.jpg" alt="Figure 4"/> 
Figure 4. After completion of the disinfection and rinse steps of the protocol, the tubing is removed and the heart is placed on an absorbent pad to allow the excess water to drain out of the heart. This ensures an accurate measurement when weighing the tissue and also allows the tissue to relax before sectioning.
<img src="/files/ftp_upload/50059/50059fig5.jpg" alt="Figure 5"/> 
Figure 5. The left ventricle (LV), right ventricle (RV), and ventricular septum are all removed from the decellularized heart for histologic processing, freezing and lyophilization, and DNA quantification.
<img src="/files/ftp_upload/50059/50059fig6.jpg" alt="Figure 6" fo:content-width="4.5in" fo:src="/files/ftp_upload/50059/50059fig6highres.jpg"/> 
Figure 6. Quantitative analysis of DNA content using a Pico Green assay. The ventricles from cECM hearts show a significant decrease in DNA content when compared to native ventricles. The DNA values observed from this protocol are observed at or below the 50 ng/mg standard for decellularized tissues.
<img src="/files/ftp_upload/50059/50059fig7.jpg" alt="Figure 7"/> 
Figure 7. DNA fragment size, as determined by ethidium bromide gel, showed little residual DNA in the decellularized ventricles when compared to a urinary bladder matrix (UBM) standard.
<img src="/files/ftp_upload/50059/50059fig8.jpg" alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/50059/50059fig8highres.jpg"/> 
Figure 8. Hematoxylin and Eosin staining showed complete removal of nuclear material from the ventricles following completion of the decellularization protocol.
<img src="/files/ftp_upload/50059/50059fig9.jpg" alt="Figure 9" fo:content-width="3in" fo:src="/files/ftp_upload/50059/50059fig9highres.jpg"/> 
Figure 9. Masson's Trichrome staining of A) native and B) decellularized ventricle.

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

23.8K Views.  McGowan Institute for Regenerative Medicine. 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.

Video: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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 ...

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

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, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

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 ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

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 ...

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.

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 ...

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 ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.