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

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

<ol>
	<li>Writing Group Members. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Executive+Summary:+Heart+Disease+and+Stroke+Statistics--2016+Update:+A+Report+From+the+American+Heart+Association.">Executive Summary: Heart Disease and Stroke Statistics--2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), 447-454 (2016).</li><li>Zia, S., 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=Hearts+beating+through+decellularized+scaffolds:+whole-organ+engineering+for+cardiac+regeneration+and+transplantation.">Hearts beating through decellularized scaffolds: whole-organ engineering for cardiac regeneration and transplantation.</a> Critical Reviews in Biotechnology. 36 (4), 705-715 (2016).</li><li>Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strip+and+Dress+the+Human+Heart.">Strip and Dress the Human Heart.</a> Circulation Research. 118 (1), 12-13 (2016).</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> Nature Medicine. 14 (2), 213-221 (2008).</li><li>Sanchez, P. L., 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=Acellular+human+heart+matrix:+A+critical+step+toward+whole+heart+grafts.">Acellular human heart matrix: A critical step toward whole heart grafts.</a> Biomaterials. 61, 279-289 (2015).</li><li>Peloso, 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=Current+achievements+and+future+perspectives+in+whole-organ+bioengineering.">Current achievements and future perspectives in whole-organ bioengineering.</a> Stem Cell Research &amp; Therapy. 6, 107 (2015).</li><li>Guyette, J. 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=Perfusion+decellularization+of+whole+organs.">Perfusion decellularization of whole organs.</a> Nature Protocols. 9 (6), 1451-1468 (2014).</li><li>Momtahan, N., Sukavaneshvar, S., Roeder, B. L., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+Processes+to+Decellularize+and+Cellularize+Hearts+to+Generate+Functional+Organs+and+Reduce+the+Risk+of+Thrombosis.">Strategies and Processes to Decellularize and Cellularize Hearts to Generate Functional Organs and Reduce the Risk of Thrombosis.</a> Tissue Engineering Part B-Reviews. 21 (1), 115-132 (2015).</li><li>Lee, P. F., 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=Inverted+orientation+improves+decellularization+of+whole+porcine+hearts.">Inverted orientation improves decellularization of whole porcine hearts.</a> Acta Biomaterialia. , (2016).</li><li>Momtahan, N., 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=Automation+of+Pressure+Control+Improves+Whole+Porcine+Heart+Decellularization.">Automation of Pressure Control Improves Whole Porcine Heart Decellularization.</a> Tissue Eng Part C Methods. , (2015).</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. 75 (4), 852-860 (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=Bioartificial+heart:+A+human-sized+porcine+model--the+way+ahead.">Bioartificial heart: A human-sized porcine model--the way ahead.</a> PLoS One. 9 (11), e111591 (2014).</li><li>Remlinger, N. T., Wearden, P. D., 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=Procedure+for+decellularization+of+porcine+heart+by+retrograde+coronary+perfusion.">Procedure for decellularization of porcine heart by retrograde coronary perfusion.</a> Journal of Visualized Experiments. (70), e50059 (2012).</li><li>Guyette, J. 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=Bioengineering+Human+Myocardium+on+Native+Extracellular+Matrix.">Bioengineering Human Myocardium on Native Extracellular Matrix.</a> Circulation Research. 118 (1), 56-72 (2016).</li><li>Sanchez, P. L., 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=Data+from+acellular+human+heart+matrix.">Data from acellular human heart matrix.</a> Data Brief. 8, 211-219 (2016).</li><li>Garreta, 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=Myocardial+commitment+from+human+pluripotent+stem+cells:+Rapid+production+of+human+heart+grafts.">Myocardial commitment from human pluripotent stem cells: Rapid production of human heart grafts.</a> Biomaterials. 98, 64-78 (2016).</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 Engineering Part C-Methods. 16 (3), 525-532 (2010).</li><li>Larson, A. M., Yeh, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+of+sub-10-fs+pulses.">Ex vivo characterization of sub-10-fs pulses.</a> Optics Letters. 31 (11), 1681-1683 (2006).</li><li>Lee, P. F., Yeh, A. T., Bayless, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+microscopy+reveals+invading+endothelial+cells+anisotropically+alter+three-dimensional+collagen+matrices.">Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices.</a> Experimental Cell Research. 315 (3), 396-410 (2009).</li><li>Lee, P. F., Bai, Y., Smith, R. L., Bayless, K. J., Yeh, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+responses+are+enhanced+in+mechanically+and+microscopically+characterized,+microbial+transglutaminase+crosslinked+collagen+matrices+with+increased+stiffness.">Angiogenic responses are enhanced in mechanically and microscopically characterized, microbial transglutaminase crosslinked collagen matrices with increased stiffness.</a> Acta Biomaterialia. 9 (7), 7178-7190 (2013).</li><li>Wu, Z., 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=Multi-photon+microscopy+in+cardiovascular+research.">Multi-photon microscopy in cardiovascular research.</a> Methods. 130, 79-89 (2017).</li><li>Ramanathan, T., Skinner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+blood+flow.">Coronary blood flow.</a> Continuing Education in Anaesthesia Critical Care &amp; Pain. 5 (2), 61-64 (2005).</li><li>Murthy, V. L., 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=Clinical+Quantification+of+Myocardial+Blood+Flow+Using+PET:+Joint+Position+Paper+of+the+SNMMI+Cardiovascular+Council+and+the+ASNC.">Clinical Quantification of Myocardial Blood Flow Using PET: Joint Position Paper of the SNMMI Cardiovascular Council and the ASNC.</a> Journal of Nuclear Cardiology. 25 (1), 269-297 (2018).</li><li>Molina, D. K., DiMaio, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+organ+weights+in+men:+Part+I-the+heart.">Normal organ weights in men: Part I-the heart.</a> The American Journal of Forensic Medicine and Pathology. 33 (4), 362-367 (2012).</li><li>Molina, D. K., DiMaio, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+Organ+Weights+in+Women:+Part+I-The+Heart.">Normal Organ Weights in Women: Part I-The Heart.</a> The American Journal of Forensic Medicine and Pathology. 36 (3), 176-181 (2015).</li><li>Robertson, M. J., Dries-Devlin, J. L., Kren, S. M., Burchfield, J. S., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+cellularization+of+whole+decellularized+heart+extracellular+matrix.">Optimizing cellularization of whole decellularized heart extracellular matrix.</a> PLoS One. 9 (2), e90406 (2014).</li><li>Robertson, M. J., Soibam, B., O'Leary, J. G., Sampaio, L. C., Taylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellularization+of+rat+liver:+An+in+vitro+model+for+assessing+human+drug+metabolism+and+liver+biology.">Cellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.</a> PLoS One. 13 (1), e0191892 (2018).</li><li>Baghalishahi, 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=Cardiac+extracellular+matrix+hydrogel+together+with+or+without+inducer+cocktail+improves+human+adipose+tissue-derived+stem+cells+differentiation+into+cardiomyocyte-like+cells.">Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte-like cells.</a> Biochemical and Biophysical Research Communications. , (2018).</li><li>Perea-Gil, I., 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=In+vitro+comparative+study+of+two+decellularization+protocols+in+search+of+an+optimal+myocardial+scaffold+for+recellularization.">In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization.</a> American Journal of Translational Research. 7 (3), 558-573 (2015).</li><li>Freytes, D. O., O'Neill, J. D., Duan-Arnold, Y., Wrona, E. A., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+cardiac+extracellular+matrix+hydrogels+for+cultivation+of+human+stem+cell-derived+cardiomyocytes.">Natural cardiac extracellular matrix hydrogels for cultivation of human stem cell-derived cardiomyocytes.</a> Methods Molecular Biology. 1181, 69-81 (2014).</li><li>Oberwallner, B., 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+scaffolds+by+decellularization+of+human+myocardium.">Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium.</a> Journal of Biomedical Materials Research Part A. 102 (9), 3263-3272 (2014).</li></ol>

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

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

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

<table><tbody><tr><td>2-0 silk suture</td><td>Ethicon</td><td>SA85H</td><td>Suture used to ligate superior and inferior vena cava</td></tr><tr><td>1/4&quot; x 3/8&quot; 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...

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

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

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

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Research

JoVE Journal

Bioengineering

11.2K Views.  Texas Heart Institute. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over het genereren van gevasculariseerde steigers, die echt de heilige graal van het weefsel engineering veld. Het belangrijkste voordeel van deze techniek is dat een onder druk gestelde zak in een omgekeerde oriëntatie de decellularisatie van niet-transplanteerbare menselijke organen verbetert en ons dat op een steriele manier over een geschikt tijdsbestek laat doen. Om het hart voor te bereiden op decellularisatie, voert u...

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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering 

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

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

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

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

Cancer Research

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Biology

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Comprehensive 3D Analysis of Human Hearts from Micro to Macro Scales

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results&#160;to illustrate progress in the research of human cardiac anatomy.

Author Spotlight: Advancing Human Cardiac Anatomy Through Multi-Scale Analysis of Hearts

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic ...

Whole Organ Tissue Culture

Whole organs can be cultured ex vivo using specialized bioreactors&#160;with the goal of repairing or replacing entire organs. This method uses a donor organ that is stripped of all cells, leaving behind the three-dimensional structure, which&#160;is then repopulated with new cells. This video demonstrates the whole organ culture of lungs&#160;and shows how a dynamic culture that mimics the mechanical stimulation in the body is needed to induce native tissue properties.

Whole organs can be cultured&#160;ex vivo&#160;using specialized bioreactors&#160;with the goal of repairing or replacing entire organs. This method uses a donor organ that is stripped of all cells, leaving behind the three-dimensional structure, which&#160;is then repopulated with new cells. This video demonstrates the whole organ culture of lungs&#160;and shows how a dynamic culture that mimics the mechanical stimulation in the body is needed to induce native tissue properties.

Whole organs can be cultured ex vivo using specialized bioreactors&#160;with the goal of repairing or replacing entire organs. This method uses a donor ...

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

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