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. 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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. R., 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=Molecular+and+biomechanical+clues+from+cardiac+tissue+decellularized+extracellular+matrix+drive+stromal+cell+plasticity.">Molecular and biomechanical clues from cardiac tissue decellularized extracellular matrix drive stromal cell plasticity.</a> Front Bioeng Biotechnol. 8, 520 (2020).</li><li>Silva, A. C., Pereira, C., Fonseca, A. C. R. G., Pinto-do-&#211;, P., Nascimento, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bearing+my+heart:+The+role+of+extracellular+matrix+on+cardiac+development,+homeostasis,+and+injury+response.">Bearing my heart: The role of extracellular matrix on cardiac development, homeostasis, and injury response.</a> Front Cell Dev Biol. 8, 621644 (2021).</li></ol>

<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

إنتاج مصفوفة القلب خارج الخلية من الخلايا الليفية البشرية البالغة لطلاء طبق الثقافة

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

390 Views.  University of Salerno. في البروتوكول ، نصف طريقة مجدية للحصول على مصفوفة القلب خارج الخلية في المختبر كركيزة لدراسة تفاعلات مصفوفة الخلية وتأثير المصفوفة خارج الخلية الخاصة بعضلة القلب على خلايا القلب. تركز خطوط البحث الحالية على تطوير دعامات ثلاثية الأبعاد مهندسة بيولوجيا م...

Video: Author Spotlight: Studying Cardiac Cell-Matrix Interactions In Vitro

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. After decellularization, this substrate can be used for the culture and study of other cardiac cells and cell-matrix interactions.

Culturing Human Cardiac Fibroblasts from Atrial Myocardium Fragments

To begin, wash the fresh human atrium sample in a 100 millimeter glass plate containing sterile HBSS. Gently shake the samples in the plate to remove any residual blood. Transfer the sample to a 100 millimeter plate wetted with a small volume of HBSS.With crossed scalpels, chop the sample into two-millimeter-sized cubes. Next, transfer four small myocardial fragments to a 35 millimeter plate. Place a sterile cover glass over the fragments and apply gentle pressure.Pipette 1.5 milliliters of DMEM into the plate. Then incubate the culture plates at 37 degrees Celsius, under 5%carbon dioxide supplementation. When the cultures have reached 85%confluency, remove the plate from the incubator and use sterile forceps to lift the cover glass.Then place it upside down on a new 35 millimeter plate. Then add sterile PBS to rinse it. Now remove the myocardium fragments.Then pipette DMEM out of the plate. Use sterile PBS to rinse the plate containing the outgrown cells. After discarding the rinse, add one milliliter of 0.25%trypsin EDTA to each plate, and incubate the plates at 37 degrees Celsius, with 5%carbon dioxide, for five minutes.After incubation, remove the plates from the incubator and use a microscope to confirm the cell detachment. Next, add two milliliters of trypsin stop solution, or TSS, into each plate to block the trypsinization. Transfer the cell suspension into a sterile 15 milliliter tube.Centrifuge it at 400 G for five minutes at four degrees Celsius. Aspirate the supernatant using a five milliliter serological pipette. Then add three milliliters of DMEM to the pellet and resuspend it.Place the cell suspension on a 60 millimeter plate. Incubate the cells at 37 degrees Celsius, under 5%carbon dioxide supplementation. Next, pipette three milliliters of sterile 0.2%gelatin solution to each 60 millimeter plate.After incubation, aspirate the solution and add three milliliters of sterile PBS until further use. Take the plate containing cardiac fibroblasts out of the incubator. Remove DMEM from the plate and rinse with sterile PBS.After discarding the rinse, add one milliliter of 0.25%trypsin EDTA to each plate. Use a microscope to confirm the cell detachment. Next, add two milliliters of TSS into each plate To block the trypsinization.Transfer the cell suspension into a sterile 15 milliliter tube. Centrifuge it at 400 G for five minutes at four degrees Celsius. Aspirate the supernatant using a five milliliter serological pipette.Then add three milliliters of DMEM to the pellet and resuspend it. Remove the PBS from the 60 millimeter gelatin-coated plate and place the cell suspension into the plate. Culture the fibroblasts in a confluence state for up to 21 days.After 21 days of culture, aspirate the culture medium. Then rinse the plates with three milliliters of PBS. After aspirating the PBS and adding one milliliter of decellularization solution, observe the plate under an inverted contrast microscope.After two minutes, gently pipette four milliliters of PBS to dilute the decellularization solution in the plates. Aspirate the diluted solution. Then gently rinse the plates with PBS.Finally, add one milliliter of PBS to the plates. Store the extracellular matrix-coated plates at four degrees Celsius until further use. The outgrowth of fibroblasts from the small fragments of native myocardium placed in culture was observed within three to five days.Decellularization resulted in extracellular matrix coating on the plate surface. The composition and architecture of the in vitro-produced cardiac extracellular matrix was preserved due to decellularization.