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

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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. 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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. 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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 substrate for in vitro cell culture has attracted the interest of biologists, leading to several attempts at developing such a substrate using fibroblasts isolated from neonatal animal hearts. As early as 1996, Vanwinkle et al.13 used neonatal rat cardiac fibroblasts to produce extracellular matrix coating on glass coverslips using EDTA, a calcium chelating agent, for cell separation. More recently, EDTA has been used in combination with a basic solution of Triton X-100 at lower concentrations14. Calcium ions mediate interactions between cells and extracellular matrix, as well as between different extracellular matrix molecules15. However, using such chelating agents can lead to matrix protein unfolding and loss of interactions between its different fibrillar and amorphous components, inevitably modifying the cardiac ECM obtained in vitro. In our experience, incubation with 0.5 mM EDTA required a lengthy time, with the first signs of cell detachment observable at the inverted phase-contrast microscope not appearing until after 2 h.
A similar method for in vitro decellularization was published by Hellewell et al.16 However, in the present protocol, a lower concentration of ammonium hydroxide (10 mM instead of 20 mM) was used, and Triton X-100 was added as a decellularization agent. While the authors of the mentioned protocol tested its application in three commercially available cell types, primitive cardiac fibroblasts were not included. Given the longstanding difficulty in obtaining and culturing adult human cardiomyocytes or cardiac fibroblasts in vitro, the focus here was on cardiac tissue and cells, which are of primary interest to cardiovascular research groups and cardiovascular regenerative medicine in general. It is possible that this solution may be suitable for other cell types as well.
While denaturation is necessary for protein solubilization, the use of a non-denaturing decellularization agent is essential for the quantitative and qualitative preservation of extracellular matrix components for further in vitro use. Therefore, Triton X-100 was used instead of SDS to remove the fibroblasts, leaving the intact cardiac ECM coating on the surface of the Petri dish. Incubation with a basic solution of Triton X-100 induces membrane permeabilization and cell lysis, resulting in fibroblast removal. If nuclear debris is an issue (for example, when subsequent experiments require cell transfection or nucleic acid molecular analysis), it is possible to minimize DNA content with DNase I before seeding other cell types on cardiac ECM.
The most significant advantage of the method detailed here is the ability to obtain cardiac ECM typical of different conditions related to human heart structure and function, as the same fibroblasts influenced by those changing conditions in vivo deposit the extracellular matrix in vitro. Thus, 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 functional interactions in various normal and pathological conditions17,18.
Although it may be tempting to stimulate fibroblast activity in vitro by adding ascorbic acid, known to stimulate the production of collagen and glycosaminoglycans, such selective stimulation is discouraged, as it could potentially modify the composition of the obtained cardiac ECM towards a fibrotic phenotype19. Instead, depending on the study&#39;s aim, the composition of the cardiac ECM can be influenced in vitro by the addition of factors such as angiotensin peptides or adrenaline20,21, known to play a crucial role in the pathogenesis and pathophysiology of specific cardiovascular disorders.
The critical step in the process of extracellular matrix deposition, followed by fibroblast layer removal from culture, is the adhesion and preservation of fibroblast-derived matrix deposited on the dish surface. Unlike previously discussed studies where neonatal fibroblasts were cultured for only a few days, adult human cardiac fibroblasts survive in culture in the confluent state for as long as 21 days, continuously synthesizing and secreting extracellular matrix. It is essential to use gelatin-coated culture dishes and visually control fibroblast detachment, reducing the time of incubation in the decellularization solution to the necessary minimum.
In the present protocol, a feasible method for obtaining cardiac extracellular matrix in vitro is described. The 2D monolayer system reflects the in vivo microenvironment thanks to the presence of the native extracellular matrix produced by the same cells that are responsible for extracellular matrix synthesis and maintenance, namely fibroblasts, with the composition influenced by the biological cues present in vivo. Other biomimetic and 3D cell culture substrates or systems, such as hydrogels, spheroids, organoids, and 3D bioprinting methods, have attracted considerable attention as a strategy to mimic in vivo conditions, allowing the study of the complex spatial cell-cell and cell-ECM interactions22,23. These approaches address the spatial and biomechanical cues for in vitro cell growth and differentiation24,25, but they present strong limitations with regard to the composition and characteristics of the produced substrates, which hardly resemble those of the native tissue. Based on the scope of the study and its applications, the 2D cardiac ECM or 3D biomimetic scaffolds can be used, and together, they represent a complementary method for enhancing the current knowledge of cell biology and the capabilities of tissue regeneration.
Limitations 
The method described is subject to certain limitations, particularly concerning the decellularization process designed to eliminate cellular elements. This process could inadvertently disturb the natural architecture or composition of the ECM. However, the ECM obtained from normal and pathological heart-derived fibroblasts differently influences the proliferation, apoptosis, and migration of cultured cells11,18, indicating that the described method preserves at least the main determinants of these biological processes.
Similarly, cardiac ECM obtained from different donors may have different compositions, even if the main cardiac pathology is the same. As with all human samples, there is great variability resulting from environmental factors, genetic determinants, pharmacological or other therapies, among others26. The cardiac ECM obtained by the described method should not always be expected to have the same composition and characteristics. If qualitative reproducibility is essential, it may be worth considering expanding the isolated fibroblasts up to two times (as described in the present protocol) to produce ECM on numerous Petri dishes or using fibroblasts obtained from different donors in the same Petri dish for ECM production.

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 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

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 number of fibroblasts within the same time, as the resulting tissue fragments can have slightly different volumes and cell survival rates. Notably, myocardium fragments with confirmed outgrowth activity can be retained and replated, thus allowing them to be used at least twice for fibroblast isolation.
The fibroblasts can be subcultured for several passages to increase the number of cells available for further experiments. However, once they reach confluence, fibroblasts cease to proliferate in vitro and assume synthesis and secretion activity, depositing extracellular matrix. Subsequent decellularization leaves the surface of the plate coated with cardiac ECM mesh (Figure 2). Visual observation of the culture dish, with the naked eye and using an inverted phase-contrast microscope, during fibroblast removal is essential to minimize the time of incubation in the decellularization solution to the necessary and sufficient minimum. The presence of a floating veil during incubation indicates permeabilization and detachment of the monolayer of fibroblasts, and the decellularization solution should be diluted and removed immediately after.
The composition of the fibroblast-derived cardiac ECM depends on the source of the fibroblasts. In our experiments, the cardiac ECM produced by fibroblasts isolated from normal human hearts and from human hearts with ischemic cardiopathy had different protein contents and exerted different influences on primitive cardiac cell proliferation, apoptosis, and migration11,12. Although ECM protein loss is inevitable during the decellularization process, the method employed in the present protocol preserved the composition and architecture of the in vitro produced cardiac ECM to a large extent (Figure 3).
In this protocol, atrial tissue was used because it can be obtained from both normal (donor) and pathological (recipient) hearts. While the whole pathological (recipient) heart can be sectioned and used for research (following the principles of the Declaration of Helsinki and with local ethical committee approval), it is not possible to obtain samples of ventricles from the donor heart at the time of transplantation. For this reason, if the scope of the study is to investigate differences between cardiac tissue in normal and pathological conditions, the best source of sample is the atrial wall. Depending on the availability of other viable cardiac tissue sources, samples from atrial or ventricular walls of the normal or pathological hearts can be used following the same protocol for cardiac ECM dish coating.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66160/66160fig01.jpg" /> 
Figure 1: Isolation of cardiac fibroblasts. Fibroblast outgrowth from myocardium fragments after 4 days (A), 8 days (B), and 12 days (C) days of culture. Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66160/66160fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66160/66160fig02.jpg" /> 
Figure 2: Decellularization of extracellular matrix. Fibroblasts after approximately 10 s (A), 20 s (B), 30 s (C), 40 s (D), 50 s (E), and 60 s (F) of incubation in the decellularization solution. Scale bars: 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66160/66160fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66160/66160fig03.jpg" /> 
Figure 3: Extracellular matrix composition before and after decellularization. Indirect immunofluorescence staining of representative cardiac extracellular matrix proteins, including type III (A,E) and type IV (B,F) collagen, laminin (C,G), and fibronectin (D,H), before (A-D) and after (E-H) fibroblast removal. Cell nuclei were stained with DAPI (blue). Scale bars: (A-C,E-G) 50 &#181;m; (D,H) 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66160/66160fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solutions</td>
			<td colspan="2">Composition of solutions&#160;</td>
			<td>Final volume</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td colspan="2">Commercially powered salts + double-distilled sterile water + 0.35 g sodium bicarbonate</td>
			<td>1 L</td>
			<td>Sterilized by filtration/store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Decellularization solution</td>
			<td colspan="2">0.25% Triton X-100 + 10 mM NH4OH+ PBS</td>
			<td>50 mL</td>
			<td>Sterilized by filtration/store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td colspan="2">223.75 mL DMEM + 25 mL fetal bovine serum + 1.25 mL penicillin/streptomycin solution</td>
			<td>250 mL</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Gelatin solution&#160;</td>
			<td colspan="2">0.2 g gelatin powder + 100 mL Phosphate-buffered-saline&#160;</td>
			<td>100 mL</td>
			<td>Sterilized by filtration/store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Phosphate-buffered-saline (1x PBS)</td>
			<td colspan="2">0.1 g potassium phosphate monobasic + 0.1 g potassium chloride + 4.0 g sodium chloride + 0.575 g sodium phosphate dibasic + double-distilled sterile water</td>
			<td>500 mL</td>
			<td>Sterilized by filtration/store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypsin stop solution (TSS)</td>
			<td colspan="2">5 mL fetal bovine serum + 45 mL Hank&#39;s Balanced Salt Solution</td>
			<td>50 mL</td>
			<td>Store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: The components and the respective concentrations for each solution utilized in the study.

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

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. ...

production-cardiac-extracellular-matrix-from-adult-human-fibroblasts

Nanyang Technological University

Research

JoVE Journal

Biology

545 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

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

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

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Chapters in this video

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart