Name: comparable decellularization of fetal and adult cardiac tissue explants as 3d-like platforms for in vitro studies
Uploaded: 2019-03-21T12:30:00
Description: Watch this Scientific Journal Video about Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies at JoVE.com

The authors are indebted to all members of Pinto-do-&#211; laboratory for relevant critical discussion. This work was supported by Programa MIT-Funda&#231;&#227;o para Ci&#234;ncia e Tecnologia (FCT) under the project &#34;CARDIOSTEM-Engineered cardiac tissues and stem cell-based therapies for cardiovascular applications&#34; (MITP-TB/ECE/0013/2013). A.C.S. is a recipient of a FCT fellowship [SFRH/BD/88780/2012] and M.J.O. is a FCT Fellow (FCT-Investigator 2012).

All the methodologies described were approved by the i3S Animal Ethics Committee and Dire&#231;&#227;o Geral de Veterin&#225;ria (DGAV) and are in accordance with the European Parliament Directive 2010/63/EU.
1. Preparation of the decellularization solutions
NOTE: All decellularization solutions should be filtered through a 0.22 &#956;m membrane filter and stored for a maximum of 3 months, except specified otherwise.
<ol>
	<li>For 1x PBS: Mix 8 g of NaCl, 1.01 g o...

<ol>
	<li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> Journal of Cell Science. 123 (24), 4195-4200 (2010).</li><li>Rozario, T., DeSimone, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+in+development+and+morphogenesis:+a+dynamic+view.">The extracellular matrix in development and morphogenesis: a dynamic view.</a> Dev Biol. 341 (1), 126-140 (2010).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Organ+Tissue+Engineering:+Decellularization+and+Recellularization+of+Three-Dimensional+Matrix+Scaffolds.">Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds.</a> Annu Rev Biomed Eng. 13 (1), 27-53 (2011).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14 (2), 213-221 (2008).</li><li>Silva, A. 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=Three-dimensional+scaffolds+of+fetal+decellularized+hearts+exhibit+enhanced+potential+to+support+cardiac+cells+in+comparison+to+the+adult.">Three-dimensional scaffolds of fetal decellularized hearts exhibit enhanced potential to support cardiac cells in comparison to the adult.</a> Biomaterials. 104, 52-64 (2016).</li><li>Nakayama, K. H., Batchelder, C. A., Lee, C. I., Tarantal, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+rhesus+monkey+kidney+as+a+three-dimensional+scaffold+for+renal+tissue+engineering.">Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.</a> Tissue Eng Part A. 16 (7), 2207-2216 (2010).</li><li>Williams, C., Quinn, K. P., Georgakoudi, I., Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Young+developmental+age+cardiac+extracellular+matrix+promotes+the+expansion+of+neonatal+cardiomyocytes+in+vitro.">Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro.</a> Acta Biomater. 10 (1), 194-204 (2014).</li><li>Fong, A. 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=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 Eng Part A. 22 (15-16), 1016-1025 (2016).</li><li>Tapias, L. F., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+scaffolds+as+a+platform+for+bioengineered+organs.">Decellularized scaffolds as a platform for bioengineered organs.</a> Curr Opin Organ Transplant. 19 (2), 145-152 (2014).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27 (19), 3675-3683 (2006).</li><li>Pinto, M. 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=Decellularized+human+colorectal+cancer+matrices+polarize+macrophages+towards+an+anti-inflammatory+phenotype+promoting+cancer+cell+invasion+via+CCL18.">Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.</a> Biomaterials. 124, 211-224 (2017).</li><li>Garlikova, 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=Generation+of+a+close-to-native+in+vitro+system+to+study+lung+cells-ECM+crosstalk.">Generation of a close-to-native in vitro system to study lung cells-ECM crosstalk.</a> Tissue Eng Part C: Methods. , (2017).</li><li>Wong, M. L., Griffiths, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+in+xenogeneic+scaffold+generation:+Antigen+removal+vs.+decellularization.">Immunogenicity in xenogeneic scaffold generation: Antigen removal vs. decellularization.</a> Acta Biomaterialia. 10 (5), 1806-1816 (2014).</li><li>Motsavage, V. A., Kostenbauder, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+the+state+of+aggregation+on+the+specific+acid-catalyzed+hydrolysis+of+sodium+dodecyl+sulfate.">The influence of the state of aggregation on the specific acid-catalyzed hydrolysis of sodium dodecyl sulfate.</a> J Colloid Sci. 18 (7), 603-615 (1963).</li><li>Rahman, A., Brown, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+the+critical+micelle+concentration+of+sodium+dodecyl+sulphate.">Effect of pH on the critical micelle concentration of sodium dodecyl sulphate.</a> J Appl Polymer Sci. 28 (4), 1331-1334 (1983).</li><li>Brown, B. N., 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+as+an+inductive+scaffold+for+functional+tissue+reconstruction.">Extracellular matrix as an inductive scaffold for functional tissue reconstruction.</a> Transl Res. 163 (4), 268-285 (2014).</li><li>Ieda, 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+fibroblasts+regulate+myocardial+proliferation+through+beta1+integrin+signaling.">Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.</a> Dev Cell. 16 (2), 233-244 (2009).</li><li>Whitby, D. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+of+lip+wounds+in+fetal,+neonatal+and+adult+mice.">The extracellular matrix of lip wounds in fetal, neonatal and adult mice.</a> Development. 112 (2), 651-668 (1991).</li><li>Brennan, E. P., Tang, X. H., Stewart-Akers, A. M., Gudas, 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=Chemoattractant+activity+of+degradation+products+of+fetal+and+adult+skin+extracellular+matrix+for+keratinocyte+progenitor+cells.">Chemoattractant activity of degradation products of fetal and adult skin extracellular matrix for keratinocyte progenitor cells.</a> J Tissue Eng Regen Med. 2 (8), 491-498 (2008).</li><li>Coolen, N. A., Schouten, K. C., Middelkoop, E., Ulrich, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+human+fetal+and+adult+skin.">Comparison between human fetal and adult skin.</a> Arch Dermatol Res. 302 (1), 47-55 (2010).</li></ol>

The extracellular matrix (ECM) is a highly dynamic and complex meshwork of fibrous and adhesive glycoproteins, consisting of a reservoir of numerous bioactive peptides and entrapped growth factors. As the major modulator of cell adhesion, cytoskeleton dynamics, motility/migration, proliferation, differentiation and apoptosis, ECM actively regulates cellular function and behavior. Knowing that cellular behavior differs in 2D and 3D cultures, there have been efforts to develop novel organotypic models that can accurately r...

An erratum was issued for:&#160;<a href="https://www.jove.com/video/56924/comparable-decellularization-fetal-adult-cardiac-tissue-explants-as">Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies</a>.&#160; The&#160;affiliations were&#160;updated.
The fourth&#160;affiliation&#160;was corrected from:
Gladstone Institutes, University of California San Francisco
to:
Gladstone Institute&#160;of Cardiovascular Disease
An additional affiliation was added for Todd C. McDevitt:
University of California San Francisco

An erratum was issued for:&#160;<a href="https://www.jove.com/video/56924/comparable-decellularization-fetal-adult-cardiac-tissue-explants-as">Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies</a>.&#160; An affiliation&#160;was updated.

Erratum: Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

The extracellular matrix (ECM) is a dynamic network of molecules that regulate important cellular processes, namely fate-decision, proliferation and differentiation1,2. The investigation of cell-ECM interactions has been performed mainly in two-dimensional (2D) in vitro cultures coated with ECM components, which constitute a simplification of native ECM properties found in vivo. Decellularization generates acellular 3D-like ECM bioscaffolds that largely preserve the extracellular architecture and composition of native tissues and organs3,4. In addition to serving as bioactive scaffolds for tissue engineering, decellularized 3D ECM biomaterials are emerging as novel platforms to assess cell-ECM biology that parallel the in vivo environment.
Assessment of the differential role of the ECM components of distinct tissues, organs and age will benefit with the use of similar protocols of generating native bioscaffolds. In the heart, we have developed a versatile protocol for decellularization of fetal and adult-derived samples, as an alternative approach to perform comparative studies of the organ microenvironment. Using this methodology, we captured the native cardiac microenvironment and showed that fetal ECM promotes higher repopulation yields of cardiac cells5. Decellularization further provided identification of resident structural differences between fetal and adult ECM at the level of basement lamina and pericellular matrix mesh arrangement and fiber composition5. Prior to this work, head-to-head comparison of tissues at different ontogenic stages using the same decellularization approach has only been reported for rhesus monkey kidneys and rodent hearts. In addition, a limited number of studies report fetal tissue/organ decellularization per se5,6,7. This has been achieved using SDS as a unique decellularization agent; however, distinct SDS concentrations were used for the decellularization of fetal and adult cardiac tissue7,8. SDS is one of the most effective ionic detergents for clearance of cytoplasmic and nuclear material, and widely used in the decellularization of different tissues and specimens9,10. Solutions containing high SDS concentrations and extended periods of exposure have been correlated with protein denaturation, glycosaminoglycan (GAGs) loss and disruption of collagen fibrils10,11, and therefore a balance between ECM preservation and cell removal is necessary. To apply the same procedure to fetal and adult heart tissue, the protocol described herein is divided in three sequential steps: cell lysis by osmotic shock (hypotonic buffer); solubilization of lipid-protein, DNA-protein and protein-protein interactions (0.2% SDS); and nuclear material removal (DNase treatment).
Our protocol shows several advantages: i) the possibility of equivalent decellularization of age-specific cardiac tissues by the application of the same decellularization strategy; ii) no requirements for specialized methods or equipment; iii) ready adaptation to other tissues and species as it has been successfully applied with minor alterations in human intestine biopsies12 and mouse lung13; and, importantly, iv) can address ECM biomechanical properties while enabling the assembly of 3D-like organotypic cultures that more closely mimic the molecular features of the native tissue microenvironment.

<table>
	<tbody>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Incubated Benchtop Shaker</td>
			<td>Orbital Shakers</td>
			<td>IKA:3510001</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Fluorimeter</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Digital weight scale</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Fridge (4&#186;C)</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Deep freezer (-80&#186;C)</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>-</td>
			<td>-</td>
			<td>Equipment available</td>
		</tr>
		<tr>
			<td colspan="4">Cirurgical Instruments</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>5000-08</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Dumont 5 Fine Forceps - Biologie/Inox</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Dumont 7 forceps</td>
			<td>Fine Science Tools</td>
			<td>11272-30</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>Dissecting Scissors, straight</td>
			<td>-</td>
			<td>-</td>
			<td>Tool available</td>
		</tr>
		<tr>
			<td>Forceps, serrated, curved</td>
			<td>-</td>
			<td>-</td>
			<td>Tool available</td>
		</tr>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>24 well plates, individually wrapped</td>
			<td>VWR</td>
			<td>29442-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>96 well plates, individually wrapped</td>
			<td>VWR</td>
			<td>71000-078</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Steriflip-GV, 0.22&#181;m, PVDF, Radio-Sterilized</td>
			<td>Millipore</td>
			<td>SE1M179M6</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eppendorff</td>
			<td>-</td>
			<td>-</td>
			<td>Material available</td>
		</tr>
		<tr>
			<td>15 mL Falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>430791</td>
			<td>-</td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Fisher Scientific</td>
			<td>430829</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Four-Compartment Biopsy Processing/Embedding Cassettes with Lid</td>
			<td>Electron Microscopy Science</td>
			<td>70075-B</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-037-246</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Tissue cryopreservation</td>
		</tr>
		<tr>
			<td>Shandon Cryomatrix embedding resin</td>
			<td>Thermo Scientific</td>
			<td>6769006</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-METHYLBUTANE ANHYDROUS 99+% (isopentane)</td>
			<td>Sigma-Aldrich</td>
			<td>277258-1L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Decellularization</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BDH Prolabo</td>
			<td>27810.364</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S-31264</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-100g</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P8041-1KG</td>
			<td>-</td>
		</tr>
		<tr>
			<td>TrisBASE</td>
			<td>Sigma-Aldrich</td>
			<td>T6066-500G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L-4390</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>MERCK</td>
			<td>1.05833.1000</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>AplliChem</td>
			<td>A3778,0050</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-049</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Gibco BRL</td>
			<td>15290-026</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Deionized water (DI water)</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Histology</td>
		</tr>
		<tr>
			<td>10 % formalin neutral buffer</td>
			<td>Prolabo</td>
			<td>361387P</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eosin Y AQUEOUS</td>
			<td>Surgipath</td>
			<td>01592E</td>
			<td>Can be replaced by alcoholic eosin</td>
		</tr>
		<tr>
			<td>Richard-Allan Scientific HistoGel Specimen Processing Gel</td>
			<td>Thermo Fisher Scientific</td>
			<td>HG-4000-012</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethanol ethilic alcohol 99,5% anydrous</td>
			<td>Aga</td>
			<td>4,006,02,02,00</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Deionized water (DI water)</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Clear Rite 3</td>
			<td>Richard-Allan Scientific</td>
			<td>6915</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Shandon Histoplast</td>
			<td>Thermo Fisher Scientific</td>
			<td>RAS.6774006</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Kits</td>
		</tr>
		<tr>
			<td>PureLink Genomic DNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K182001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen&#160;dsDNA kit</td>
			<td>Invitrogen</td>
			<td>P11496</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>VWR</td>
			<td>45000-434</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100X</td>
			<td>Labclinics</td>
			<td>L0022-100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Gibco BRL</td>
			<td>15290-026</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell culture media of the cell of interest</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

comparable decellularization of fetal and adult cardiac tissue explants as 3d-like platforms for in vitro studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The decellularization efficiency should be assessed through three main techniques: macroscopic observation, histology and DNA quantification. The macroscopic appearance of samples post-SDS treatment indirectly affects the efficacy of cell removal. After SDS incubation, samples should appear as translucent to whitish (Figure 1C). Fetal (E18) decellularized tissues are characterized by a highly translucent structure while adult explants have a translucent to wh...

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Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

This method can help address important questions in the cardiovascular field about the impact the extracellular matrix has during different physiological and developmental stages of the heart. The main advantage of this technique is that it allows a comparative analysis between fetal and adult extracellular matrices by applying a similar decellularization method to both types of tissue. So this method have been successful applied to other organs such as surgical resections from cancer patients facilitating the study of the extracellular matrix in different contexts.Begin by briefly rinsing the fetal and adult mouse hearts with ice-cold PBS. Then place the tissues under a dissecting microscope and use straight small scissors to remove the atria, right ventricle, and the septa from the adult mouse hearts. Transfer the left ventricle to a new Petri dish and divide the left ventricle free wall into two millimeter thick longitudinal strips.The use of adult mouse left ventricle explants of a homogeneous size is essential for a consistent decellularization efficiency. Use the scalpel and serrated tweezers with curved tip to remove the papillary muscle before using a two by two millimeter grid to further mince the strips into smaller tissue fragments. When all of the tissue has been minced, briefly rinse the pieces with enough PBS to cover the explants.And transfer the fetal hearts and pieces of adult heart tissue into individual 1.5 milliliter microcentrifuge tubes containing 250 microliters of optimal cutting temperature or OCT compound per tube. Then, freeze the cardiac tissue samples in dry ice cooled isopentane for storage at negative 80 degrees Celsius. To decellularize the cryo-preserved tissues place the frozen samples in a Petri dish containing enough PBS to completely cover the explants.After the OCT has melted, submerge the samples in a new Petri dish of PBS and wash the samples three times on a shaker for 10 to 15 minutes at 60 RPM per wash. After the last wash, add one milliliter of working hypotonic buffer to each well of a sterile 24-well tissue culture plate and use fine forceps to transfer one fragment to each well. Incubate the plate at 25 degrees Celsius for 18 hours, at 165 RPM followed by three one-hour washes in one milliliter of PBS per well, per wash.After the last wash, add one milliliter of freshly prepared sodium dodecyl sulfate or SDS solution to each well for a 24-hour incubation at 25 degrees Celsius. The next day, rinse the samples with three 20 minute washes in one milliliter of hypotonic wash buffer per wash followed by the addition of one milliliter of DNase treatment solution to each well for a three-hour incubation, at 37 degrees Celsius. At the end of the incubation, the decellularized samples should lose their gelatin-like consistency.Then wash the tissue fragments three times with one milliliter of PBS for 20 minutes per wash followed by an overnight wash in one milliliter of fresh PBS per well at room temperature and 60 RPM. To assess the removal of cellular remnants from the decellularized tissue, the next morning fix the samples in one milliliter of freshly prepared 10%formula neutral buffer supplemented with 0.03%of aqueous eosin per well for 2.5 to three hours at room temperature. At the end of the incubation, wash the samples in one milliliter of PBS per well.And use a disposable vinyl specimen mold to encapsulate the decellularized fragments in histology processing gel according to the manufacturer's specifications. Next, transfer the encapsulated fragments to a biopsy processing embedding cassette. And process the samples for paraffin embedding through successive 30-minute incubations in ascending concentrations of ethanol, isoparaffinic aliphatic hydrocarbon solution and two 56 degrees Celsius paraffin emergens.After the second paraffin emergent, mount samples in a paraffin block and use a microtome to obtain three-micrometer thick sections. Then verify the decellularization efficiency by staining the sections with haematoxylin and eosin and Masson's trichrome stains according to standard protocols. After the overnight PBS wash with shaking, add 500 microliters of freshly prepared DPBS, supplementd with 2.5 micrograms per milliliter of amphotericin B 1%antibiotic solution to each scaffold.And store the samples for one to seven days at four degrees Celsius. Before seating the cells, replace the DPBS solution with 500 microliters of cell basal medium supplemented with antibiotics. And incubate the scaffolds for one hour at 37 degrees Celsius.At the end of the incubation, use a microscope to add a four microliter drop of DPBS to the center of one well of a 96-well plate per scaffold. And use thin straight tweezers to transfer one decellularized scaffold to the top of each drop. Remove any extra DPBS by aspiration and confirm that the scaffold is not folded or wrinkled.When the scaffolds have dried at the edges, slowly seat a single cell suspension of the cells of interest on the top of each scaffold. Embryonic day 18 decellularized tissues are characterized by a highly translucent structure while adult explants exhibit a translucent to white appearance. Hematoxylin and eosin and Masson's trichome stains confirm an efficient cell removal by the presence porous mesh.The nuclear material is reduced by approximately 99.8%after decellularization, compared to non-manipulated tissues which is essential for avoiding triggering an undesired inflammatory response upon implantation. The cell viability within seated scaffolds can be monitored during in-vitro culture by calcein staining. Terminal analysis of the cell repopulation and distribution across scaffolds, can also be performed after paraffin processing as observed in these representative images of a central bioscaffold section.So this technique paved the way for researchers in the exploration of the bioactive properties of the extracellular matrix from fetal and adult mouse hearts but also of other tissues.

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