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>
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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. 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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. ...

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