Generation of Myocardium-Mimicking Extra Cellular Matrix for Cell Culture

<ol>
	<li>Gattazzo, F., Urciuolo, A., Bonaldo, P. <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:+A+dynamic+microenvironment+for+stem+cell+niche.">Extracellular matrix: A dynamic microenvironment for stem cell niche.</a> Biochim Biophys Acta. 1840 (8), 2506-2519 (2014).</li><li>Zhang, Y., 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=Tissue-specific+extracellular+matrix+coatings+for+the+promotion+of+cell+proliferation+and+maintenance+of+cell+phenotype.">Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.</a> Biomaterials. 30 (23-24), 4021-4028 (2009).</li><li>Xing, H., Lee, H., Luo, L., Kyriakides, T. R. <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-derived+biomaterials+in+engineering+cell+function.">Extracellular matrix-derived biomaterials in engineering cell function.</a> Biotechnol Adv. 42, 107421 (2020).</li><li>Andr&#233;s-Delgado, L., Mercader, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+cardiac+function+and+heart+development.">Interplay between cardiac function and heart development.</a> Biochim Biophys Acta. 1863 (7), 1707-1716 (2016).</li><li>Hall, C., Gehmlich, K., Denning, C., Pavlovic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+relationship+between+cardiac+fibroblasts+and+cardiomyocytes+in+health+and+disease.">Complex relationship between cardiac fibroblasts and cardiomyocytes in health and disease.</a> J Am Heart Assoc. 10 (5), e019338 (2021).</li><li>Castaldo, C., Chimenti, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+progenitor+cells:+The+matrix+has+you.">Cardiac progenitor cells: The matrix has you.</a> Stem Cells Transl Med. 7, 506-510 (2018).</li><li>Nurzynska, D., Iruegas, M. E., Castaldo, C., M&#252;ller-Best, P., Di Meglio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+biotechnology+in+myocardial+regeneration-tissue+engineering+triad:+cells,+scaffolds,+and+signaling+molecules.">Application of biotechnology in myocardial regeneration-tissue engineering triad: cells, scaffolds, and signaling molecules.</a> Biomed Res Int. 2013, 236893 (2013).</li><li>Giacca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+after+myocardial+infarction:+An+approachable+goal.">Cardiac regeneration after myocardial infarction: An approachable goal.</a> Curr Cardiol Rep. 22 (10), 122 (2020).</li><li>Spedicati, 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=Biomimetic+design+of+bioartificial+scaffolds+for+the+in+vitro+modeling+of+human+cardiac+fibrosis.">Biomimetic design of bioartificial scaffolds for the in vitro modeling of human cardiac fibrosis.</a> Front Bioeng Biotechnol. 10, 983782 (2022).</li><li>Souders, C. A., Bowers, S. L., Baudino, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast:+The+renaissance+cell.">Cardiac fibroblast: The renaissance cell.</a> Circ Res. 105 (12), 1164-1176 (2009).</li><li>Castaldo, 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+fibroblast-derived+extracellular+matrix+(biomatrix)+as+a+model+for+the+studies+of+cardiac+primitive+cell+biological+properties+in+normal+and+pathological+adult+human+heart.">Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.</a> Biomed Res Int. 2013, 352370 (2013).</li><li>Nurzynska, D., 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+produced+cardiac+extracellular+matrix+for+studies+of+myocardium+regeneration+potential.">In vitro produced cardiac extracellular matrix for studies of myocardium regeneration potential.</a> Tissue Eng Part A. 21, 77 (2015).</li><li>VanWinkle, W. B., Snuggs, M. B., Buja, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+ECM:+A+biosynthetic+extracellular+matrix+for+cardiomyocyte+culture.">Cardiac ECM: A biosynthetic extracellular matrix for cardiomyocyte culture.</a> In Vitro Cell Dev Biol Anim. 32, 478-485 (1996).</li><li>Santhakumar, R., Vidyasekar, P., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+ECM:+A+nano-matrix+scaffold+with+potential+application+in+cardiac+regeneration+using+mesenchymal+stem+cells.">Cardiac ECM: A nano-matrix scaffold with potential application in cardiac regeneration using mesenchymal stem cells.</a> PLoS One. 9 (12), e114697 (2014).</li><li>Gopal, S., Multhaupt, H. A. B., Couchman, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+in+cell-extracellular+matrix+interactions.">Calcium in cell-extracellular matrix interactions.</a> Adv Exp Med Biol. 1131, 1079-1102 (2020).</li><li>Hellewell, A. L., Rosini, S., Adams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid,+scalable+method+for+the+isolation,+functional+study,+and+analysis+of+cell-derived+extracellular+matrix.">A Rapid, scalable method for the isolation, functional study, and analysis of cell-derived extracellular matrix.</a> J Vis Exp. (119), e55051 (2017).</li><li>Nurzynska, D., Di Meglio, F., Montagnani, S., Castaldo, C., Hayat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+stem+cells+derived+from+epithelial-mesenchymal+transition+of+epicardial+cells:+role+in+heart+regeneration+(method).">Cardiac stem cells derived from epithelial-mesenchymal transition of epicardial cells: role in heart regeneration (method).</a> Stem Cells and Cancer Stem Cells, vol. 5: Therapeutic Applications in Disease and Injury. , 109-115 (2012).</li><li>Pagano, 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=Normal+versus+pathological+cardiac+fibroblast-derived+extracellular+matrix+differentially+modulates+cardiosphere-derived+cell+paracrine+properties+and+commitment.">Normal versus pathological cardiac fibroblast-derived extracellular matrix differentially modulates cardiosphere-derived cell paracrine properties and commitment.</a> Stem Cells Int. 2017, 7396462 (2017).</li><li>Xu, Y., 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=Vitamin+C+regulates+the+profibrotic+activity+of+fibroblasts+in+in+vitro+replica+settings+of+myocardial+infarction.">Vitamin C regulates the profibrotic activity of fibroblasts in in vitro replica settings of myocardial infarction.</a> Int J Mol Sci. 24 (9), 8379 (2023).</li><li>Jaffr&#233;, 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=Serotonin+and+angiotensin+receptors+in+cardiac+fibroblasts+coregulate+adrenergic-dependent+cardiac+hypertrophy.">Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy.</a> Circ Res. 104 (1), 113-123 (2009).</li><li>B&#322;yszczuk, 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=Activated+cardiac+fibroblasts+control+contraction+of+human+fibrotic+cardiac+microtissues+by+a+&#946;-adrenoreceptor-dependent+mechanism.">Activated cardiac fibroblasts control contraction of human fibrotic cardiac microtissues by a &#946;-adrenoreceptor-dependent mechanism.</a> Cells. 9 (5), 1270 (2020).</li><li>Duval, K., 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=Modeling+physiological+events+in+2D+vs.+3D+cell+culture.">Modeling physiological events in 2D vs. 3D cell culture.</a> Physiol. 32 (4), 266-277 (2017).</li><li>Ungerleider, J. 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=Fabrication+and+characterization+of+injectable+hydrogels+derived+from+decellularized+skeletal+and+cardiac+muscle.">Fabrication and characterization of injectable hydrogels derived from decellularized skeletal and cardiac muscle.</a> Methods. 84, 53-59 (2015).</li><li>Seo, Y., 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+heart+ECM+hydrogel+using+supercritical+carbon+dioxide+for+improved+angiogenesis.">Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis.</a> Acta Biomater. 67, 270-281 (2018).</li><li>Liguori, G. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L laboratory bottle&#160;</td>
			<td>VWR</td>
			<td>215-1595</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>Sterile,&#160; polystyrene</td>
		</tr>
		<tr>
			<td>100 mm glass plate&#160;</td>
			<td>VWR</td>
			<td>391-0578</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>100 mm plates</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>15 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>22 mm x 22 mm cover glasses</td>
			<td>VWR</td>
			<td>631-1570</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>Falcon</td>
			<td>357525</td>
			<td>Sterile,&#160; polystyrene</td>
		</tr>
		<tr>
			<td>250 mL laboratory bottle&#160;</td>
			<td>VWR</td>
			<td>215-1593</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>35 mm plates</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>5 mL serological pipet</td>
			<td>Falcon</td>
			<td>357543</td>
			<td>Sterile, polystyrene</td>
		</tr>
		<tr>
			<td>50 mL sterile tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Centrifuge sterile tubes, polypropylene</td>
		</tr>
		<tr>
			<td>500 mL laboratory bottle</td>
			<td>VWR</td>
			<td>215-1594</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>60 mm plates</td>
			<td>Falcon</td>
			<td>353004</td>
			<td>Treated, sterile cell culture dish</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide (NH4OH)</td>
			<td>Sigma- Aldrich</td>
			<td>338818</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>Disposable scalpels</td>
			<td>VWR</td>
			<td>233-5526</td>
			<td>Sterile and disposable</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Sigma- Aldrich</td>
			<td>D6429-500ml</td>
			<td>Store at 2-8 &#176;C; avoid exposure to light</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma- Aldrich</td>
			<td>F9665-500ml</td>
			<td>Store at -20 &#176;C. The serum should be aliquoted into smaller working volumes</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>VWR</td>
			<td>232-1317</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma- Aldrich</td>
			<td>G1890-100G</td>
			<td>Commercial Powder</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Sigma- Aldrich</td>
			<td>H1387-1L</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>VWR</td>
			<td>233-1211</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>Microdissecting scissors&#160;</td>
			<td>Sigma- Aldrich</td>
			<td>S3146</td>
			<td>Clean and autoclave before use</td>
		</tr>
		<tr>
			<td>Penicillin and Streptomycin&#160;</td>
			<td>Sigma- Aldrich</td>
			<td>P4333-100ml</td>
			<td>Store at -20&#176;C. The solution&#160; should be aliquoted into smaller working volumes</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma- Aldrich</td>
			<td>P9333</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma- Aldrich</td>
			<td>P5665</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Chloride&#160;</td>
			<td>Sigma- Aldrich</td>
			<td>S7653</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma- Aldrich</td>
			<td>94046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Stericup Filters</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>Sterile and disposable 0.22 mm filter membranes&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma- Aldrich</td>
			<td>9002-93-1</td>
			<td>Liquid</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma- Aldrich</td>
			<td>T4049-100ml</td>
			<td>Store at -20 &#176;C. It should be aliquoted into smaller working volumes</td>
		</tr>
	</tbody>
</table>

production of cardiac extracellular matrix from adult human fibroblasts for culture dish coating

All cells are located in vivo in a specialized microenvironment in which they can survive and carry out their specific functions. Within any given tissue, the cells are surrounded by an extracellular matrix composed of fibrillar and non-fibrillar proteins, and fundamental substances rich in glycosaminoglycans1. The qualitative and quantitative changes in the matrix content influence cell biology, controlling processes such as cell proliferation, apoptosis, migration, or differentiation. Hence, efforts are invested in recreating this microenvironment for in vitro studies of cells from different tissues2,3.
The myocardium consists of cardiomyocytes and an even larger quantity of fibroblasts that play a critical role in producing and maintaining the extracellular matrix within the myocardium4. Throughout life, the composition of the extracellular matrix can change in response to various normal and pathological factors. These modifications in extracellular matrix composition have a significant impact on the structure and biomechanical characteristics of the myocardium5. Accordingly, it should be advantageous for understanding cell-matrix interactions within the human myocardium if the microenvironment specific to different ages or pathological conditions was reproduced in vitro6,7.
The method described here aims to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo.
Cardiovascular research presents specific challenges, including the difficulty in obtaining samples from living donors or patients and culturing human cardiac cells8. The method presented here addresses these challenges by enabling the acquisition of cardiac fibroblasts even from small bioptic fragments of human myocardium and culturing isolated cardiac cells in vitro on their native extracellular matrix typical of the human myocardium.
While current efforts focus on developing 3D scaffolds of biofartificial synthetic or natural polymers that mimic the biomechanical properties of normal myocardium9, they overlook the cell-matrix interactions and signaling that occur in both normal and pathological conditions. Since the cardiac ECM is synthesized by cardiac fibroblasts derived from the human heart, its composition is determined by the activity of these cells, which changes in response to various physiological and pathological conditions, thereby allowing the study of its specific influence on cardiac cell biology10.
The current protocol was specifically designed for human cardiac tissue, but its scientific basis should also apply to other organs, especially those with low regeneration potential, intense fibrosis, and scarring influencing overall structure and function, as well as limited sample numbers and sizes.

Author Spotlight: Studying Cardiac Cell-Matrix Interactions In Vitro

从成人成纤维细胞中生产心脏细胞外基质，用于培养皿包被

In the protocol, we describe a feasible method to obtain the cardiac extracellular matrix in vitro as a substrate for studying the cell-matrix interactions and the influence of myocardium-specific extracellular matrix on cardiac cells. Current lines of research focus on development of bioengineered, three-dimensional supports made of biomimetic materials that can be seeds with cells. This material seem to provide biomechanical and spatial cues for cells.The composition of extracellular matrix has a profound impact on cells, with the bi-directional signaling taking place and leading to continuous modifications in both matrix composition and cell behavior. In vitro models reproducing cardiac microenvironment, typical of physiological or pathological conditions are missing. A better understanding of cell matrix interactions offered by the ability to recreate them in vitro can significantly improve the design of biomimetic scaffolds for tissue engineering and regeneration.

Read this Scientific Article Protocol about Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating at JoVE.com

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. ...

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Biology

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating

356 次观看.  University of Salerno. 在该方案中，我们描述了一种可行的方法，可以在体外获得心脏细胞外基质作为研究细胞 - 基质相互作用和心肌特异性细胞外基质对心肌细胞影响的底物。目前的研究方向集中在开发由仿生材料制成的生物工程三维支架上，这些支架可以是带有细胞的种子。这种材料似乎为细胞提供了生物力学和空间线索。细胞外基质的组成对细胞有深远的影响...