Name: processing of human cardiac tissue toward extracellular matrix self-assembling hydrogel for in vitro and in vivo applications
Uploaded: 2017-12-04T14:00:00
Description: Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki. Patients provided informed consent for the use of the tissue for research purposes, and the process of tissue collection was approved by the Institutional Review Board and ethics committee of Charit&#233; - Universit&#228;tsmedizin Berlin (EA4/028/12).

The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Charit&#233; Medical University. All patients provided written, informed consent for use of heart tissue for experimental studies.
1. Preparation of Human Myocardium for Sectioning
<ol>
	<li>Obtain left ventricular myocardium (size varies depending on the type of surgery) directly from the operating room and transport in cold...

<ol>
	<li>Elliott, R., Hoehn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Commercial+Porcine+Skin+for+Wound+Dressings.">Use of Commercial Porcine Skin for Wound Dressings.</a> Plastic and reconstructive surgery. 52 (4), 401-405 (1973).</li><li>Rienks, M., Papageorgiou, A. -. P., Frangogiannis, N. G., Heymans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+Extracellular+Matrix:+An+Ever-Changing+and+Diverse+Entity.">Myocardial Extracellular Matrix: An Ever-Changing and Diverse Entity.</a> Circulation Research. 114 (5), 872-888 (2014).</li><li>Prabhu, S. D., Frangogiannis, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biological+Basis+for+Cardiac+Repair+After+Myocardial+Infarction.">The Biological Basis for Cardiac Repair After Myocardial Infarction.</a> Circulation Research. 119 (1), 91-112 (2016).</li><li>Boopathy, A. V., Martinez, M. D., Smith, A. W., Brown, M. E., Garcia, A. J., Davis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+Delivery+of+Notch+Ligand-Containing+Hydrogels+Improves+Cardiac+Function+and+Angiogenesis+Following+Infarction.">Intramyocardial Delivery of Notch Ligand-Containing Hydrogels Improves Cardiac Function and Angiogenesis Following Infarction.</a> Tissue Eng Part A. 21 (17-18), 2315-2322 (2015).</li><li>Gaetani, R., Yin, 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=Cardiac+derived+extracellular+matrix+enhances+cardiogenic+properties+of+human+cardiac+progenitor+cells.">Cardiac derived extracellular matrix enhances cardiogenic properties of human cardiac progenitor cells.</a> Cell Transplant. , (2015).</li><li>Kraehenbuehl, T. P., Ferreira, L. 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=Human+embryonic+stem+cell-derived+microvascular+grafts+for+cardiac+tissue+preservation+after+myocardial+infarction.">Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction.</a> Biomaterials. 32 (4), 1102-1109 (2011).</li><li>Zhang, J., Klos, 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=Extracellular+matrix+promotes+highly+efficient+cardiac+differentiation+of+human+pluripotent+stem+cells:+The+matrix+sandwich+method.">Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: The matrix sandwich method.</a> Circulation Research. 111 (9), 1125-1136 (2012).</li><li>Fong, A. H., Romero-L&#243;pez, 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=Three-Dimensional+Adult+Cardiac+Extracellular+Matrix+Promotes+Maturation+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes.">Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.</a> Tissue Engineering Part A. 22 (15-16), 1016-1025 (2016).</li><li>DeQuach, J. A., Mezzano, V., 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=Simple+and+High+Yielding+Method+for+Preparing+Tissue+Specific+Extracellular+Matrix+Coatings+for+Cell+Culture.">Simple and High Yielding Method for Preparing Tissue Specific Extracellular Matrix Coatings for Cell Culture.</a> PLoS ONE. 5 (9), e13039 (2010).</li><li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., 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=Extracellular+matrix+hydrogels+from+decellularized+tissues:+Structure+and+function.">Extracellular matrix hydrogels from decellularized tissues: Structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li><li>Tukmachev, D., Forostyak, 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=Injectable+extracellular+matrix+hydrogels+as+scaffolds+for+spinal+cord+injury+repair.">Injectable extracellular matrix hydrogels as scaffolds for spinal cord injury repair.</a> Tissue Eng Part A. , (2016).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., 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=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Singelyn, J. M., Sundaramurthy, 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=Catheter-deliverable+hydrogel+derived+from+decellularized+ventricular+extracellular+matrix+increases+endogenous+cardiomyocytes+and+preserves+cardiac+function+post-myocardial+infarction.">Catheter-deliverable hydrogel derived from decellularized ventricular extracellular matrix increases endogenous cardiomyocytes and preserves cardiac function post-myocardial infarction.</a> Journal of the American College of Cardiology. 59 (8), 751-763 (2012).</li><li>Wainwright, J. M., Czajka, C. 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=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 (3), 525-532 (2010).</li><li>Ott, H. C., Matthiesen, T. 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=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, 213-221 (2008).</li><li>Oberwallner, B., Anic, B. 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=Human+cardiac+extracellular+matrix+supports+myocardial+lineage+commitment+of+pluripotent+stem+cells.">Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells.</a> Eur J Cardiothorac Surg. 47, 416-425 (2015).</li><li>Oberwallner, B., Brodarac, 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=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><li>Kappler, B., Anic, 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+cytoprotective+capacity+of+processed+human+cardiac+extracellular+matrix.">The cytoprotective capacity of processed human cardiac extracellular matrix.</a> Journal of Materials Science: Materials in Medicine. , (2016).</li><li>Bashey, R. I., Martinez-Hernandez, A., Jimenez, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+localization+of+cardiac+collagen+type+VI.+Associations+with+other+extracellular+matrix+components.">Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.</a> Circulation Research. 70 (5), (1992).</li><li>Wu, J., Ravikumar, P., Nguyen, K. T., Hsia, C. C. W., Hong, Y., Gorler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+protection+by+inhalation+of+exogenous+solubilized+extracellular+matrix.">Lung protection by inhalation of exogenous solubilized extracellular matrix.</a> PLOS ONE. 12 (2), e0171165 (2017).</li><li>Chen, W. C. W., Wang, 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=Decellularized+zebrafish+cardiac+extracellular+matrix+induces+mammalian+heart+regeneration.">Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration.</a> Science Advances. 2 (11), e1600844 (2016).</li><li>Godier-Furn&#233;mont, A. F. G., Martens, T. 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=Composite+scaffold+provides+a+cell+delivery+platform+for+cardiovascular+repair.">Composite scaffold provides a cell delivery platform for cardiovascular repair.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7974-7979 (2011).</li><li>Sarig, U., Sarig, 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=Natural+myocardial+ECM+patch+drives+cardiac+progenitor+based+restoration+even+after+scarring.">Natural myocardial ECM patch drives cardiac progenitor based restoration even after scarring.</a> Acta Biomaterialia. 44, 209-220 (2016).</li><li>Singelyn, J. M., DeQuach, J. A., Seif-Naraghi, S. B., Littlefield, R. B., Schup-Magoffin, P. J., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30 (29), 5409-5416 (2009).</li></ol>

When preparing human myocardial ECM, the goal is to achieve the following: removal of relevant immunogenic cellular material, preservation of ECM integrity and bioactivity, sterility, non-toxicity of the end-product, GMP-process compatibility, and suitability of the product for a given application in terms of handling. By combining our 3-step decellularization protocol with further processing into microparticles or self-assembling hydrogel, human cardiac ECM material is obtained that possesses specific biological activit...

The extracellular matrix (ECM) provides not only structural support but is also important for biologic cell and tissue function1. In the heart, the ECM participates in the regulation of pathophysiologic responses such as fibrosis, inflammation, angiogenesis, cardiomyocyte contractile function and viability, and resident progenitor cell fate. In addition to its primary components - fibrous glycoproteins, glycosaminoglycans, and proteoglycans - it contains a host of secreted growth factors, cytokines, and membranous vesicles containing nucleic acids and proteins2,3.
It has recently become clear that acellular ECM preparations are not only invaluable for studying cell-matrix interactions, but also for potential therapeutic cell-based applications. The importance of providing an adequate environment to therapeutic cell products or engineered tissues is now widely acknowledged. Attempts have been made to combine cell suspensions or active compounds with defined biopolymeric hydrogels4,5,6 or with protein cocktails secreted by murine sarcoma cells (i.e., Matrigel, Geltrex)7. However, the former have limited bioactivity, the latter are problematic in GMP-grade processes, and both lack the tissue-specific bioactivity of cardiac ECM (cECM)8,9,10,11,12,13.
Decellularization of the myocardium has previously been performed by perfusion of the whole heart via the coronary vasculature14,15. While this is possible in animal hearts, intact human hearts are rarely available. Therefore, an immersion process that allows for handling tissue samples obtained in the operating room was favored. Our &#34;3-step&#34; protocol contains 3 separate incubation steps namely lysis, solubilization, and DNA removal. It yields human myocardial ECM with largely preserved protein and glycosaminoglycan composition16,17. These cECM slices allow for in vitro studies of cell-matrix interactions but are poorly suited for potential human-scale therapeutic applications. The manufacturing process was then extended to produce either lyophilized cECM microparticles or a cECM hydrogel18.
This protocol allows for the decellularization of human myocardium obtained from surgical samples, preserving the major components of myocardial extracellular matrix (ECM) and their biologic activity. This protocol is recommended when human cardiac ECM with preserved tissue-specific bioactivity is required for experimental studies of cell-matrix interactions, or when a suitable environment is needed for cell-based myocardial regeneration approaches. In principle, it is also possible to adapt this protocol to GMP-grade conditions, so that the use of processed cECM should be feasible in future therapeutic applications.

<table>
	<tbody>
		<tr>
			<td>Balance</td>
			<td>DR Precisa, Dietikon, Switzerland</td>
			<td>Precisa XR 205SM</td>
			<td></td>
		</tr>
		<tr>
			<td>Blades Nr.10 Skalpell Nr.3</td>
			<td>InstrumenteNRW, Erftstadt, Germany</td>
			<td>SK-10-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (6-well)</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat CM</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8043.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf reaction tubes (1.5 or 2 ml)</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>616201, 623201</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15ml, 50ml</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>188271, 227270</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochrome, Berlin, Germany</td>
			<td>S 0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze Dry System</td>
			<td>Labconco, Kansas City, USA</td>
			<td>7670520</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80&#176;C)</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>Forma 900 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>281.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Blades Type 819</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>14035838925</td>
			<td></td>
		</tr>
		<tr>
			<td>Minilys Homogeniser</td>
			<td>PEQLAB Biotechnologie GmbH, Erlangen, Germany</td>
			<td>91-PCSM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>K021.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P06-07800</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>P6887-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys Keramik-Kit 1.4 mm</td>
			<td>Peqlab Biotechnolgie, Erlangen, Germany</td>
			<td>91-PCS-CK14</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotamax 120 Plate shaker</td>
			<td>Heidolph, Schwabach, Germany</td>
			<td>544-41200-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>CN30.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP, 0,22 &#181;m</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stuart analogue rocker &#38; roller mixers</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z675113-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek O.C.T compound</td>
			<td>Hartenstein, Wurzburg, Germany</td>
			<td>TTEK</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfusion set 200&#181;m</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>798.200.500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>5429.3</td>
			<td></td>
		</tr>
		<tr>
			<td>vedena Skalpellgriff Fig. 3, Standard, 125 mm</td>
			<td>Medical Highlights, Rohrdorf, Germany</td>
			<td>CV102-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie2</td>
			<td>Scientific Industry, New York, USA</td>
			<td>SI-0256</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The 3-step protocol for decellularization of human myocardium presented here results in near-complete removal of cellular material, while preserving the key ECM components and the fibrillar structure of ECM. After decellularization, the gross removal of cells from the tissue is evident by the change in color (Figure 1A). Histological analysis with H&#38;E and Masson Trichrome stains revealed the complete absence of residual intact cells (...

Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

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.

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. ...

The overall goal of this procedure is to process cardiac extracellular matrix to a hydrogel, which enables studying the effect of the matrix in various cell based assays. This method can help to answer key questions in the cardiac field, such as how extracellular matrix affects cell behavior. The main advantage of this technique is that it facilitates the use of the extracellular matrix to do large scale experiments in vitro or in vivo.The applications of this technique can be extended towards a therapy for myocardial infarction because the matrix can provide essential survival cues. To begin the experiment, use a sterile scalpel with a number ten blade, to remove fat tissue from a previously obtained left ventricular myocardium. Cut the myocardium in cubes approximately 1 by 1 by 1 cm, using a scalpel.Next, place the cubes into a sterile 50 mL tube. Store the tubes containing the cubes at 80 degrees celsius. Take the tube containing the prepared cubes out of the freezer and transport it on ice to the cryostat.Set the object and chamber temperature of the cryostat to 15 degrees celsius. Chill empty 50 mL tubes and stamps in the chamber. Add a layer of cryosection medium onto the cold stamp, and let it freeze until it is white and solid.Add a second layer of cryosection medium, and place a myocardium tube into the second layer. Make sure the cryosection medium and myocardium are completely frozen. Then, insert the stamp with frozen myocardium into the holder and tighten all the screws.Trim the surface of the tissue until an even sectioning surface is obtained. Section the frozen myocardium with automatic sectioning to guarantee uniform cut sections. Approximately 30 to 40 sections can be sliced from one cube.Place each slice, after sectioning, loosely in a pre-cooled 50 mL tube. Close the filled tubes and place them on ice. Transfer the frozen sections of all 50 mL tubes, into one sterile beaker.Add 50 mL of lysis solution per 50 mL tube containing myocardium slices. Next, shake the myocardium slices in a sterile beaker. Strain the liquid with the tissue slices through a coarse strainer.Then, transfer the tissue slices with a blunt tweezer to a new sterile beaker. Add 50 mL of 0.5%SDS in PBS per initial 50 mL tube of myocardium slices. Shake the beaker continuously for six hours at 100-150 rpm at room temperature.After shaking, strain the liquid with the tissues slices through a coarse strainer. Using blunt tweezer, transfer the tissues slices to a new sterile beaker and wash the slices three times with 50 mL of PBS. Next, wash the slices overnight in PBS, with 1%penicillin streptomycin and 1%nystatin.The next day, remove the washing solution, and add 25 mL of FBS preheated at 37 degrees celsius with 1%penicillin streptomycin and 1%nystatin, to the decellularized slices per initial 50 mL tube of myocardium slices. Incubate the slices for three hours at 37 degrees celsius to remove any remaining DNA from the matrix. Then, transfer the slices to a new sterile beaker and wash three times with 50 mL of PBS.Place the ECM slices loosely in a new sterile six well plate. Freeze the ECM at 80 degrees celsius, and lyophilize it for two days in a lyophilize. For the pulverization of the ECM slice, use milling tubes containing 1.4 mm ceramic beads.Fill the tubes loosely with lyophilized ECM, using sterile, blunt tweezers. Snap freeze the tubes in liquid nitrogen. Insert the frozen tubes into the milling machine and pulverize the ECM.Set the duration to 30 seconds, adjust the maximum speed, and start the device. After pulverization, take out the tubes, and snap freeze them again in liquid nitrogen. Wash the microparticles from the tubes, by adding 1 mL of ddH20 per tube, and shake for complete solubilization.Then, filter the heterogeneous solution through 200 micrometer mesh, into a 50 mL tube. Freeze the tube in liquid nitrogen. Replace the standard lid of tube, with a 0.22 micrometer filter to prevent the powder from flowing out of the tube.Insert the tube in the lyophilizer, and lyophilize again to remove the water from the wash step. Dissolve the previously prepared lyophilized human ECM powder in pepsin solution, to a final concentration of 10 mg per mL. Transfer the solution to 2 mL tubes with a volume of 1 mL per tube.Pipette the solution to mix thoroughly. Next, place the tubes in the shaker and shake at 1200 rpm for 48 hours at 27 degrees celsius. Take the tubes out of the shaker and place them on ice or a pre-cooled tube holder.Mix 1/9 of the volume of the ECM solution of cold 10X PBS, with 1/10 of a volume of ECM solution of cold 0.1 molar sodium hydroxide, in a pre-cooled tube and place it on ice to neutralize the digested ECM solution and inactivate the pepsin. Finally, add the ECM solution to the neutralization mixture and mix thoroughly through pipetting or vortexing. Quantitative assays for individual ECM components revealed a more complete DNA removal and better preservation of total collagen, elastin, and glycosominoglycans as compared with decellularization by SDS, alone.RT-PCR was used to quantify the expression of structural cardiomyocite proteins, early and late cardiomyogenic transcription factors, and endothelial cells surface marker genes in Murine ESC, undergoing spontaneous differentiation. Contact with cECM drives pluripotent stem cells, preferably toward a cardiomyocite like phenotype. Phase contrast light microscopy demonstrates that enzymatic digestion of the cECM for 48 hours, results in a more homogenized ECM solution.Live/dead staining of human cardiac fibroblasts on cECM hydrogel, shows increased viability. Both cECM microparticles and cECM hydrogel enhance the metabolic activity of contractile HL-1 cells, in ischemic conditions, as compared to cells in standard culture. Once mastered, this technique can be done within one week from tissue to hydrogel, if it's performed properly.After it's development, this technique paved the way for researchers in the field of cardiac regeneration, to explore the effects of cardiac extracellular matrix, in in vitro and in vivo, model systems.

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...