Name: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion (Video) | JoVE Journal
Uploaded: 2012-12-06T12:00:00.000+00:00
Duration: 11 min 30 s
Description: Learn decellularization of porcine hearts using retrograde coronary perfusion to create cardiac extracellular matrix scaffolds. This method enables rapid removal of cellular components for tissue engineering applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">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/pubmed?term=((Perfusion-decellularized+matrix%3A+using+nature%27s+platform+to+engineer+a+bioartificial+heart%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">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/pubmed?term=((Tissue-engineered+lungs+for+in+vivo+implantation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">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/pubmed?term=((Preparation+of+cardiac+extracellular+matrix+from+an+intact+porcine+heart%5BTitle%5D)+AND+%22Tissue+Eng.+Part+C+Methods%22%5BJournal%5D)">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/pubmed?term=((An+overview+of+tissue+and+whole+organ+decellularization+processes%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Strategies+for+tissue+and+organ+decellularization%5BTitle%5D)+AND+%22Journal+of+cellular+biochemistry%22%5BJournal%5D)">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/pubmed?term=((Decellularization+of+tissues+and+organs%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((The+quest+for+an+optimized+protocol+for+whole-heart+decellularization%3A+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities%5BTitle%5D)+AND+%22Tissue+Eng.+Part+C+Methods%22%5BJournal%5D)">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/pubmed?term=((Development+and+evaluation+of+a+perfusion+decellularization+porcine+heart+model--generation+of+3-dimensional+myocardial+neoscaffolds%5BTitle%5D)+AND+%22Circulation+journal+%3A+official+journal+of+the+Japanese+Circulation+Society%22%5BJournal%5D)">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/pubmed?term=((Influence+of+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">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/pubmed?term=((Hydrated+xenogeneic+decellularized+tracheal+matrix+as+a+scaffold+for+tracheal+reconstruction%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Maintenance+of+hepatic+sinusoidal+endothelial+cell+phenotype+in+vitro+using+organ-specific+extracellular+matrix+scaffolds%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">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/pubmed?term=((Right+ventricular+outflow+tract+repair+with+a+cardiac+biologic+scaffold%5BTitle%5D)+AND+%22Cells%2C+tissues%2C+organs%22%5BJournal%5D)">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,...

<table><tbody><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>NaN&lt;sub&gt;3&lt;/sub&gt;</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

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...

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 ...

The overall goal of this procedure is to rapidly and completely remove cellular components from an intact porcine heart through retrograde profusion to yield a site-specific cardiac extracellular matrix scaffold. This is accomplished by first cannulating, the aorta of an intact porcine heart. Next tubing is attached to a peristaltic pump, and the tissue is washed with water and salt solutions at low flow rates.The heart is then perfused with a series of detergent and enzymatic solutions at a gradually increasing flow rate to remove cellular components. The final step is to perfuse a disinfectant solution. Remove the tubing and cut away the ventricular walls for processing.Ultimately, the heart is analyzed to confirm the complete removal of cellular and nuclear material from the tissue. Visual demonstration of this method is critical because the cannulation and initial profusion steps are difficult to learn because the exact position of the tubing is key to the success of the experiment. The porcine organ is obtained from an abattoir or research facility immediately after euthanasia.Begin by rinsing off any excess blood. Next, trim the heart of excess fat and tissue, keeping the atria and aorta intact. Separate the pulmonary artery from the aorta by trimming away additional fat.Once trimmed, wrap each heart individually in freezer paper and store at minus 80 degrees Celsius for at least 24 hours to ensure complete freezing. When ready to use, thaw the heart by submerging it in a four liter beaker filled with type one water and leave it at four degrees Celsius overnight. After the heart is completely thawed, pad it dry and record its weight.The heart of a market weight pig should weigh approximately 375 to 450 grams. Once the heart is ready, connect size 18 Master flex tubing to the one quarter inch end of a barbed reducer. Next, insert the barbed reducer and tubing inside the aorta.Place two 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 that the coronary arteries can be perfused. Next, use a syringe to fill the tubing with type one water and insert it into the cartridge of a master flex roller pump at its approximate midpoint.Next, submerge the inflow end of the tubing, along with the heart in the bottom of a beaker filled with 2.5 liters of water and secure. The most difficult aspects of this procedure are proper cannulation of the heart and maintenance of adequate pump speeds throughout the procedure. We ensure success in our experiment by visually inspecting the tissue for air bubbles throughout the procedure, as well as maintaining proper effusion from the ventricles throughout the procedure.Prime the pump to remove air bubbles from the tubing, reposition, or secure additional ties around the aorta and tubing if air bubbles are observed. An airtight seal is important to maintain adequate pressure during the decellularization process. At this point, warm a four liter beaker containing three liters of salt balanced trypsin solution in preparation of the decellularization process.First, set the pump to a flow rate of 400 milliliters per minute and make sure that the correct tubing size is selected. Start the pump and flush the heart with type one water for 15 to 25 minutes, the heart should swell and diffuse blood from the ventricles fresh solution should be substituted every five to 10 minutes or as needed. After the blood is removed, stop the pump and transfer the heart to a separate beaker filled with two XPBS.Submerge the tubing. Increase the flow rate to 700 milliliters per minute and start the pump. Leave the heart in solution for 15 minutes, changing the solution every five minutes.When ready, transfer the heart to type one water for 10 minutes and increase the flow rate to 750 milliliters per minute. To begin the decellularization process, transfer the heart to the beaker containing warm salt balance tryin solution. Increase the speed to 1200 milliliters per minute and start the pump.Place a stir bar at the bottom of the beaker for circulation and leave the heart in this solution for three hours. Perfusion speeds are increased slowly to condition the tissue and prevent rupture of the vessels. After one hour, increase the pump speed to 1500 milliliters per minute.After an additional hour, the pump speed is increased to 1800 milliliters per minute. The heart should swell and nearly double in size. The tissue should lose its natural color progressing from the atria to the apex.After each solution perfusion, a two step rinse is performed to remove cellular debris, chemical residue, and aid cell lysis After the salt balanced trips and solution, perfuse water at 1900 milliliters per minute and then two XPBS at 1950 milliliters per minute for 10 minutes each. Next, transfer the heart to a room temperature salt balance solution of Triton X 100. Increase the pump speed to 2000 milliliters per minute and perfuse for one hour.After refilling with fresh solution, increase the pump speed to 2100 milliliters per minute and perfuse for an additional hour and a half. Next, rinse the tissue as demonstrated before, but at increased pump speeds. The next perfusion is with room temperature 4%sodium deoxy coate.Increase the pump speed to 2200 milliliters per minute and perfuse for three hours. After three hours, repeat the rinsing steps, changing the solutions after five to 10 minutes for each solution. After the final perfusion, transfer the heart to a 0.1%paracetic acid, 4%ethanol solution and perfuse for 1.5 hours at 2200 milliliters per minute at the same pump speed, perfuse the tissue with one XPBS for 15 minutes, followed by two five minute washes in type one water.Repeat once more to complete the solution perfusion procedure, turn the pump off and remove the heart from the solution. Cut the ties from the aorta. Remove all tubing and place the heart in an empty beaker to drain for one hour.Remove excess liquid periodically after one hour. Lay the heart on an absorbent pad to drain further. After most of the water is removed, record the final weight of the heart.The heart can lose approximately 20 to 25%of its initial weight during the decellularization process. At the end of this process, the heart should appear white or translucent indicating the loss of cellular material. Next, the ventricular septum and right and left ventricles are separated and reserved for DNA.Quantification and histological processing freeze the samples at minus 80 degrees Celsius for at least two hours before lyophilization. Successful decellularization is demonstrated here by the significant decrease in DNA content when compared to native ventricles. This gel shows that little residual DNA remained in the decellularized ventricles when compared to a urinary bladder matrix or UBM.In this example, hemat toin and eosin staining shows the complete removal of nuclear material from the ventricles. Masson's staining further confirms the loss of cardiac muscle bundles and the retention of collagen networks While attempting this procedure. It is important to visually inspect the tissue and the coronaries frequently.

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

Research

JoVE Journal

Bioengineering

In this video, we perform Langendorff retrograde perfusion of an isolated mouse heart. During the procedure, the cardiac activity of the heart is maintained by perfusing it retrogradely with a perfusion solution via the coronary arteries using a cannula inserted into the ascending aorta. The model is used to study the cardiac pathophysiology of the heart.

Langendorff Retrograde Heart Perfusion: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

In this video, we describe a step-by-step protocol to extract the heart from a porcine model for cardiac transplantation.

Porcine Heart Excision: A Protocol to Extract Porcine Heart for Cardiac Transplant

This video describes an ex situ technique for preserving an isolated porcine heart by perfusing it with an oxygenated nutrient-rich perfusate solution. The technique allows the comprehensive evaluation of cardiac performance under physiological conditions.

Ex Situ Heart Perfusion: A Technique to Preserve Isolated Porcine Heart Function

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.

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

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Screening for coronary artery disease could increase the procurement of donor hearts with extended criteria. A protocol for performing reproducible coronary angiography during ex-situ heart perfusion is described herein in a porcine model.

Coronary Angiography During Ex-Situ Heart Perfusion in a Porcine Model

The protocol here describes a high-fidelity porcine model of heart transplantation following donation after circulatory death utilizing ex vivo perfusion of the allograft.

A High-Fidelity Porcine Model of Orthotopic Heart Transplantation Following Donation after Circulatory Death

Learn decellularization of porcine hearts using retrograde coronary perfusion to create cardiac extracellular matrix scaffolds. This method enables rapid removal of cellular components for tissue engineering applications.

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Explore More Videos