Cardiac tissues were obtained from patients with end-stage heart failure due to ischemic cardiopathy who were undergoing heart transplantation. All specimens used for the experiments were collected with patient consent and without patient identifiers, following the protocols approved by the ethical committee of the University of Naples Federico II and in accordance with the principles outlined in the Declaration of Helsinki. The details of all the reagents and equipment used for the study are listed in the Table of Materials.
1. Experiment preparation
<ol>
	<li>Prepare sterile tools/apparatus: One large pair of surgical scissors, two sets of fine forceps, two pairs of microdissecting scissors, 1 L sterile bottle, 500 mL sterile bottle, and a 250 mL sterile bottle.</li>
	<li>Disinfect the 22 mm x 22 mm cover glasses with 70% ethanol for a few seconds. Aspirate the ethanol using a 10 mL serological pipette, let the cover glasses dry in the oven at 37 &#176;C, and sterilize them in an autoclave.</li>
	<li>Dissolve commercially available powdered salts in sterile double-distilled water. In a 1 L sterile bottle, add 0.35 g of sodium bicarbonate powder to prepare 1 L of Hank&#39;s balanced salt solution (HBSS) at pH 7.4. Sterilize the solution using 0.22 &#181;m filter membranes under a sterile hood and store at 4 &#176;C until use.</li>
	<li>Weigh 0.1 g of potassium phosphate monobasic, 4.0 g of sodium chloride, 0.1 g of potassium chloride, and 0.575 g of sodium phosphate dibasic to prepare 500 mL of 1x phosphate-buffered saline (PBS). Dissolve the components in sterile double-distilled water and then check the pH value (7.4). Sterilize under a sterile hood by filtration and store at 4 &#176;C until use.</li>
	<li>Add 5 mL of fetal bovine serum (FBS) to 45 mL of HBSS to prepare a final volume of 50 mL of trypsin stop solution (TSS). Store at 4 &#176;C until use.</li>
	<li>Measure 223.75 mL of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 25 mL of FBS (final concentration, 10%) and add 1.25 mL penicillin and streptomycin solution (pen/strep, final concentration, 0.5%) to prepare DMEM for fibroblast isolation and culture. Store at 4 &#176;C until use.</li>
	<li>Prepare 0.2% (w/v) gelatin solution by adding 0.2 g of gelatin from porcine skin powder to 100 mL of PBS. 
	NOTE: The components and the respective concentrations for each solution utilized in the study are provided in Table 1.</li>
</ol>
2. Primitive culture of human cardiac fibroblasts by outgrowth from atrial myocardium fragments
NOTES: Steps 2.2-2.3 should be performed in sterile conditions.The protocol is validated for atrial samples approximately 0.3 cm in diameter, which is a typical dimension for biopsy specimens and allows the isolation of approximately 2 x 106 fibroblasts.
<ol>
	<li>Wash the freshly obtained sample of the atrium in a 100 mm glass plate with sterile HBSS, gently shaking it to remove the blood. Repeat this step three times, changing HBSS and the plate at each wash.</li>
	<li>Place the sample in a 100 mm plate previously wetted with a small volume of HBSS and chop finely with crossed scalpels to about 2 mm cubes.</li>
	<li>Place four small fragments of myocardium in a 35 mm plate, close enough to lie approximately at the angles of a 22 mm x 22 mm cover glass, place the sterile coverglass over the fragments, apply gentle pressure, and add 1.5 mL of DMEM.</li>
	<li>Incubate the culture plates at 37 &#176;C in 5% CO2 for about 15 days or until cells reach 85% confluence, checking daily the outgrowth of cells at an inverted phase-contrast microscope and changing the culture medium every 3 days.</li>
</ol>
3. Subculture of human cardiac fibroblasts by warm trypsinization
NOTES: The steps reported below must be performed in sterile conditions. As the fibroblasts can migrate from the explanted tissue along the surface of the cover glass and culture plate, it is recommended to process both for the first subculture.
<ol>
	<li>Lift the cover glass with fine, sterile forceps, place it upside-down in a new 35 mm plate, and rinse with sterile PBS.</li>
	<li>Remove myocardium fragments, discard them, and rinse the 35 mm plate with outgrown cells with sterile PBS.</li>
	<li>Discard the rinse and add 1 mL of 0.25% trypsin-EDTA (Ethylenediaminetetraacetic acid) to each plate. Incubate for 5 min at 37 &#176;C with 5% CO2. Confirm cell detachment at a microscope. The cells should be fully rounded up and detached, forming small clumps.</li>
	<li>Block the trypsinization by adding 2 mL of TSS to each 35 mm plate and collect the suspension into a sterile 15 mL tube, centrifuge at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant using a 5 mL serological pipette, resuspend the pellet in 3 mL of DMEM and place cell suspension into a 60 mm plate.</li>
	<li>Incubate such obtained cells in subculture 1 (or passage 1) in 3 mL of DMEM at 37 &#176;C with 5% CO2, changing the medium every three days until the cells reach 75% confluence.</li>
	<li>Subculture the fibroblasts that reached 75% confluence, repeating warm trypsinization and splitting the cells 1:3 into new 60 mm plates up to two times (passages 2 and 3).</li>
</ol>
4. Extracellular matrix deposition
<ol>
	<li>Prepare gelatin-coated dishes by adding 3 mL of sterile 0.2% gelatin solution to each 60 mm plate and incubating at 37&#160;&#176;C for 2 h; then aspirate the solution and add 3 mL of sterile PBS until use.</li>
	<li>Detach the cardiac fibroblasts by warm trypsinization and subculture them at high density (15 x 103 cells per cm2) on 60 mm gelatin-coated plates.</li>
	<li>Culture the fibroblasts in a confluent state for up to 21 days, allowing for extracellular matrix synthesis and deposition. Replace 50% of the medium with fresh medium every 3 days.</li>
</ol>
5. Extracellular matrix decellularization
NOTE: Steps 5.3-5.4 should be performed with gentle shaking to dislodge only cells without detaching the extracellular matrix.
<ol>
	<li>Prepare a decellularization solution containing 0.25% Triton X-100 and 10 mM NH4OH in PBS (calculate the final volume, considering 1 mL for every 60 mm plate).</li>
	<li>Remove the culture medium and rinse the plates with PBS. Discard the rinse.</li>
	<li>Add 1 mL of decellularization solution and observe the plates at an inverted phase-contrast microscope; after 1-2 min, when the cells are no longer discernible, gently add 4 mL of PBS to dilute the decellularization solution.</li>
	<li>Remove the diluted solution and gently rinse the plates with PBS; discard the rinse.</li>
	<li>Add 1 mL of PBS and store the ECM-coated plates at 4&#160;&#176;C until further use.</li>
</ol>

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. 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>

The authors have nothing to disclose.

The fibroblasts isolated from human heart samples were cultured to confluence for 21 days to synthesize and deposit the extracellular matrix, forming a cohesive layer firmly adherent to the surface of the culture plate. Subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of myocardium-specific extracellular matrix on other cells within the cardiac tissue.
The concept of using a natural and tissue-specific subs...

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.

<table><tbody><tr><td>1 L laboratory bottle&amp;nbsp;</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,&amp;nbsp; polystyrene</td></tr><tr><td>100 mm glass plate&amp;nbsp;</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,&amp;nbsp; polystyrene</td></tr><tr><td>250 mL laboratory bottle&amp;nbsp;</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 (NH&lt;sub&gt;4&lt;/sub&gt;OH)</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&#039;s Modified Eagle Medium (DMEM)</td><td>Sigma- Aldrich</td><td>D6429-500ml</td><td>Store at 2-8 &amp;deg;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 &amp;deg;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&#039;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&amp;nbsp;</td><td>Sigma- Aldrich</td><td>S3146</td><td>Clean and autoclave before use</td></tr><tr><td>Penicillin and Streptomycin&amp;nbsp;</td><td>Sigma- Aldrich</td><td>P4333-100ml</td><td>Store at -20&amp;deg;C. The solution&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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 &amp;deg;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

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.

The outgrowth of fibroblasts from the small fragments of native myocardium placed in culture was observed within 3-5 days (Figure 1).
In the subsequent days, the number of fibroblasts continued to increase, possibly due to sustained outgrowth from the cardiac tissue specimen and the proliferation of migrated fibroblasts on the dish surface. It should not be expected that all myocardium fragments obtained by mechanical disaggregation with a scalpel yield the same n...

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Author Spotlight: Studying Cardiac Cell-Matrix Interactions In Vitro

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.

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

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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1.1K Views.  University of Salerno. 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.

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

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

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.

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.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Bioengineering

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

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 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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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

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Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

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

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications