We thank Ms. Romy Kempe and Mrs. Annett Opitz for expert technical support. We also thank Mr. Bjoern Binnewerg for IT support. This work was supported by grants from a) the F&#246;rderkreis Dresdner Herz-Kreislauf-Tage e.V., b) &#34;Habilitationsf&#246;rderprogramm f&#252;r Frauen&#34;, Faculty of Medicine Carl Gustav Carus Dresden and c) Else Kr&#246;ner-Forschungskolleg (EKFK) Faculty of Medicine Carl Gustav Carus Dresden. We are grateful for the funding and the support.

The following protocol follows the institutional animal care guidelines of Technische Universit&#228;t Dresden, Germany (File number: T 2014/4) as well as internationally accepted animal care guidelines (FELASA)17. Figure 1 visualizes the cell isolation process.
1. Preparing the setup, material and media
<ol>
	<li>Prepare cell culture medium, PBS solution, collagenase blend stock solution (reconstitute 50 mg of lyophilized collagenase blend in 12 mL of sterile ultrapure water), and 0.25% trypsin solution.</li>
	<li>Warm up the medium, the PBS and the trypsin solution to 37 &#176;C.</li>
	<li>Preheat the ultrasonic water bath to 37 &#176;C.</li>
	<li>Disinfect forceps, a stainless-steel spatula, scalpels (2x scalpels per organ) and 2 glass beakers with 70% ethanol and place these materials under the cell culture hood.</li>
	<li>Fill one beaker with 70% ethanol and the other with sterile water or PBS solution. These beakers are required to disinfect and wash the instrument after each organ procession.</li>
	<li>Place sterile 15 mL plastic tubes containing cold PBS on wet ice. The number of tubes depends on the number of organs you want to isolate fibroblasts from.</li>
</ol>
2. Mouse dissection and organ removal
<ol>
	<li>Wear two pairs of gloves one above the other, so the first pair can be removed as soon as the animal has been dissected. 
	NOTE: This procedure prevents bacteria from the animal&#39;s fur and skin from spreading over the organs.</li>
	<li>Euthanize the mouse (e.g., cervical dislocation) and pin the carcass with needles to every limb to a polystyrene pad.</li>
	<li>Disinfect the mouse carcass using 70% ethanol spray. Make sure the fur is soaked in ethanol so the hair will not swirl up.</li>
	<li>Cut the fur right above the urogenital tract using surgical forceps and atraumatic scissors. Cut the skin alongside the middle line from the point of the initial incision to the neck (3 -&#160;4 cm) and add relief cuts at the limbs. 
	CAUTION: Do not perforate the muscular layer at this step to avoid bacterial contamination!</li>
	<li>Pin the skin to the polystyrene foam pad to have optimal access to the musculature covering the abdominal cavity.</li>
	<li>Disinfect the abdominal musculature twice using 70% ethanol. Let the ethanol dry before continuing to the next step.</li>
	<li>Remove the first pair of gloves. Use a new, sterile set of forceps and scissors.</li>
	<li>Open the abdominal cavity and the thorax by incising the muscular layer with surgical scissors to gently remove the organs of choice. Therefore, hold the organ gently with surgical forceps (do not pierce the organ, use minimal pressure) and cut the supplying blood vessels near the entry point at the organ with scissors.</li>
	<li>Put the organs into the sterile tubes containing cold PBS. Close the tubes tightly. Place the tubes on wet ice until continuing with step 3.1.</li>
</ol>
3. Tissue mincing, digestion and cell extraction
<ol>
	<li>Transfer the tubes under the sterile cell culture hood. 
	CAUTION: Wear a fresh pair of gloves and disinfect the tubes with 70% ethanol before transferring them under the hood!</li>
	<li>Take the organ out of the 15 mL tube using sterile forceps. Place the organ onto one half of a sterile 6 cm Petri dish and wash the organ briefly with PBS to remove excess blood. Transfer the organ to the second half of the Petri dish, remove excess PBS.</li>
	<li>Mince the tissue using two sterile scalpels. The remaining tissue fragments should not be larger than 1 -&#160;2 mm.</li>
	<li>Transfer the minced tissue into a new sterile 15 mL tube using the sterile spatula and add 2 mL of 0.25% trypsin solution. Place the tube into a cell culture incubator at 37 &#176;C for 5 min.</li>
	<li>Vortex the tube gently (circa 1400/min) for 10 s.</li>
	<li>Stop the trypsin reaction under the cell culture hood by adding 4 mL FCS-containing cell culture medium (Dulbecco&#39;s Modified Eagle Medium (DMEM), e.g.).</li>
	<li>Add 250 &#181;L of collagenase blend solution to each tube containing heart or lung tissue and 100 &#181;L for kidney or liver, respectively.</li>
	<li>Place the tubes into an ultrasonic water bath (37 &#176;C) and activate the ultrasonic sonicator for 10&#160;min. 
	NOTE: The ultrasonic water bath used in this protocol has an operating frequency of 35 kHz and a maximal power of 320 W.</li>
	<li>Vortex the tubes gently (circa 1400/min) for 10 s.</li>
	<li>Place the tubes again into the ultrasonic water bath for 10 min.</li>
	<li>Vortex gently (circa 1400/min) for 10 s.</li>
	<li>Disinfect the tubes with 70% ethanol and transfer them under the sterile cell culture hood.</li>
	<li>Filter the solution with a 40 &#181;m mesh into a new sterile 15 mL tube.</li>
	<li>Centrifuge the tube at 500 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 1 mL of fresh medium.</li>
	<li>Transfer the cells into a suitable cell culture vessel (e.g., 6-well plate) and place the vessel into the cell culture incubator overnight at 37 &#176;C and 5% CO2.</li>
	<li>The next day, remove the medium, wash 3 times with PBS, then add fresh medium (the added volume depends on the cell culture vessel of choice, 2 mL per well of a 6-well plate etc.).</li>
	<li>Change the medium every other day. 
	NOTE: Fibroblasts can be split after reaching optical confluence of 90% (usually after 5-7 days).</li>
</ol>

<ol>
	<li>Baum, J., Duffy, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+and+myofibroblasts:+what+are+we+talking+about.">Fibroblasts and myofibroblasts: what are we talking about.</a> Journal of Cardiovascular Pharmacology. 57 (4), 376-379 (2011).</li><li>Tallquist, M. D., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+identity+of+cardiac+fibroblasts.">Redefining the identity of cardiac fibroblasts.</a> Nature Reviews. Cardiology. 14 (8), 484-491 (2017).</li><li>Weissman-Shomer, P., Fry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+fibroblasts+senscence+in+vitro:+pattern+of+cell+division+and+life+span+as+a+function+of+cell+density.">Chick embryo fibroblasts senscence in vitro: pattern of cell division and life span as a function of cell density.</a> Mechanisms of Ageing and Development. 4 (2), 159-166 (1975).</li><li>Angello, 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=Replicative+potential+and+the+duration+of+the+cell+cycle+in+human+fibroblasts:+coordinate+stimulation+by+epidermal+growth+factor.">Replicative potential and the duration of the cell cycle in human fibroblasts: coordinate stimulation by epidermal growth factor.</a> Mechanisms of Ageing and Development. 62 (1), 1-12 (1992).</li><li>Serra, V., von Zglinicki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fibroblasts+in+vitro+senesce+with+a+donor-specific+telomere+length.">Human fibroblasts in vitro senesce with a donor-specific telomere length.</a> FEBS Letters. 516 (1), 71-74 (2002).</li><li>Mateu, 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=Functional+differences+between+neonatal+and+adult+fibroblasts+and+keratinocytes:+Donor+age+affects+epithelial-mesenchymal+crosstalk+in+vitro.">Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro.</a> International Journal of Molecular Medicine. 38 (4), 1063-1074 (2016).</li><li>Ivey, M. J., Tallquist, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+Cardiac+Fibroblast.">Defining the Cardiac Fibroblast.</a> Circulation Journal: Official Journal of the Japanese Circulation Society. 80 (11), 2269-2276 (2016).</li><li>Kanisicak, O., 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=Genetic+lineage+tracing+defines+myofibroblast+origin+and+function+in+the+injured+heart.">Genetic lineage tracing defines myofibroblast origin and function in the injured heart.</a> Nature Communications. 7, 12260 (2016).</li><li>Kuenzel, S. 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A., Bertrand-De Haas, M., Schuurman, A. H., Lamme, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzymatic+isolation+of+fibroblasts+for+the+creation+of+autologous+skin+substitutes.">Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 40 (8-9), 268-277 (2004).</li><li>El-Armouche, A., 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=Phosphatase+inhibitor-1-deficient+mice+are+protected+from+catecholamine-induced+arrhythmias+and+myocardial+hypertrophy.">Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy.</a> Cardiovascular Research. 80 (3), 396-406 (2008).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+Guidelines+and+Recommendations.">FELASA Guidelines and Recommendations.</a> Journal of the American Association for Laboratory Animal Science. 51 (3), 311-321 (2012).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments. (44), 2033 (2010).</li><li>Masur, S. K., Dewal, H. S., Dinh, T. T., Erenburg, I., Petridou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+differentiate+from+fibroblasts+when+plated+at+low+density.">Myofibroblasts differentiate from fibroblasts when plated at low density.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (9), 4219-4223 (1996).</li><li>Rohr, 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+fibroblasts+in+cell+culture+systems:+myofibroblasts+all+along.">Cardiac fibroblasts in cell culture systems: myofibroblasts all along.</a> Journal of Cardiovascular Pharmacology. 57 (4), 389-399 (2011).</li><li>Cell lines: Valuable tools or useless artifacts. PubMed - NCBI Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22553484">https://www.ncbi.nlm.nih.gov/pubmed/22553484</a> (2019)</li><li>Copp&#233;, J. 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS biology. 6 (12), 2853-2868 (2008).</li><li>Childs, B. G., Durik, M., Baker, D. J., van Deursen, J. 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There are no conflicts of interest to declare.

Compared to immortalized fibroblast cell lines, primary fibroblasts offer several advantages. They can be isolated cost effectively in high quality and quantity. Furthermore, primary cultures offer the possibility to study cells from multiple individuals, which increases the reliability of the obtained results and decreases the likelihood of merely studying cell culture artifacts. Continuous generation of new primary cultures prevents genetic alterations which commonly occur after repeated passaging21</...

Fibroblasts are flat, spindle-shaped cells with multiple stellate processes and an extensive rough endoplasmic reticulum1,2. An average fibroblast measures 30 -&#160;100 &#181;m and has a life span of 57 &#177; 3 days1,3. The average cell cycle duration of human fibroblasts ranges from 16 -&#160;48 h depending on the culture conditions4. There is evidence that the replicative capacity and functional quality of cultured primary fibroblasts negatively correlates with the donor age, suggesting that younger donors (animals or patients) should be preferred if possible5,6.
Fibroblasts constitute a predominant cell type of most mammalian body tissues. Despite their ubiquitous presence, the molecular identification of fibroblasts is still a challenge7. Fibroblasts migrate to developing tissues and organs from different sources during embryonic development8. For this reason, there is a plethora of marker proteins that can be found in fibroblasts whereas unique marker proteins, which are present in every fibroblast population and exclusive for fibroblasts, are still missing. Thus, expression patterns of several recognized markers are usually used to identify fibroblasts. Among the most recognized markers are vimentin, human fibroblast surface protein (hFSP), discoidin domain receptor 2 (DDR2) and alpha smooth muscle actin (&#945;SMA).
Fibroblasts are the major extracellular matrix (ECM)-producing cell type. Thereby, fibroblasts maintain an orderly tissue architecture and provide mechanical support for neighboring cells1. The balance between ECM synthesis and degradation is a well-regulated process. Shifts towards synthesis mark the beginning of excessive ECM deposition which, if not terminated, leads to fibrosis. Fibrosis is mediated by myofibroblasts, which originate from activated fibroblasts undergoing molecular and phenotypical changes. One hallmark of myofibroblasts is enhanced secretion of ECM and cytokines and the expression of orderly arranged &#945;SMA microfilaments9.
Primary fibroblasts have been in the spotlight of recent research focusing on fibrosis, tissue inflammation and fibroblast-cancer-cell interactions10,11. However, to effectively study fibroblast properties in health and disease, it is necessary to isolate viable primary adult fibroblasts on a regular basis. There are several methods available to isolate fibroblasts12,13,14. The three major methods of fibroblast isolation are outgrowth from tissue chunks12, enzymatic tissue digestion15, and enzymatic perfusion of hollow organs9,13,16. The advantage of outgrowth is a gentle isolation process without enzymatic cell degradation. On the other hand, outgrowth cultures usually require prolonged culture periods until cells can be used for experiments. Common enzymatic digestion is fast but bears a risk of contamination with other cell types (e.g., endothelial cells) or bacteria in the agitation process, which is necessary to mechanically dissolve the tissue. Furthermore, these methods are often elaborate and require time and skill to learn.
Regarding the importance of primary fibroblasts in research, there is still a need to optimize existing cell isolation approaches in terms of quickness, simplicity and reliability. Here, a novel ultrasonic-based enzymatic fibroblast isolation method delivering high quality cells is provided.

<table border="0" cellpadding="0" cellspacing="0" style="width:1103px;" width="1103">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:339px;">Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">T4049-100ML</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td>Gibco-Life Technologies, Carlsbad, USA</td>
			<td>Gibco LS15140148&#160;</td>
			<td>Penicillin/ Streptomycin (10000 U/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture hood</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>51023608</td>
			<td>HeraSafe KSP15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>50049176</td>
			<td>BBD 6220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plates</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td style="width:143px;">depends on vessel</td>
			<td>6-, 12-, 24-wells Nunclon surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture suction</td>
			<td style="width:339px;">VACUUBRAND GMBH + CO KG, Wertheim, Germany</td>
			<td>20727400</td>
			<td style="width:405px;">BVC professional suction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell strainer (mesh)</td>
			<td>Corning, Tewksbury, USA</td>
			<td style="width:143px;">431750</td>
			<td>40 &#181;m Nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:339px;">Thermo Fisher Scientific, Waltham, USA</td>
			<td style="width:143px;">75007213</td>
			<td>Megafuge 8R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cordless pipetting controller</td>
			<td>Hirschmann, Eberstadt, Germany</td>
			<td>9907200</td>
			<td>Pipetus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable pipette tips</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">depends on volume</td>
			<td>SafeSeal tips for pipettes (10 &#181;l, 20 &#181;l, 100 &#181;l, 200 &#181;l, 1000 &#181;l)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic pipettes</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">depends on volume</td>
			<td>5 ml, 10 ml, 25 ml, 50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpel</td>
			<td>Myco Medical, Cary, USA</td>
			<td style="width:143px;">n.a.</td>
			<td>Techno cut</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dulbeccos Modified Eagle Medium (DMEM)</td>
			<td style="width:339px;">Thermo Fisher Scientific, Waltham, USA</td>
			<td>41965-062</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf tubes</td>
			<td>Eppendorf, Hamburg, Germany&#160;</td>
			<td style="width:143px;">depends on volume</td>
			<td>50 &#181;l, 500 &#181;l, 1.500&#181;l, 2.000 &#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum (FCS)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>F2442-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase blend</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">5401020001</td>
			<td>Liberase TL Research Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish 6 cm</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P5481-500EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D8537-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Senescence detection kit</td>
			<td style="width:339px;">Abcam, Cambridge, UK</td>
			<td>ab65351</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Shaker/ Vortex</td>
			<td style="width:339px;">IKA, Staufen im Breisgau, Germany</td>
			<td style="width:143px;">n.a.</td>
			<td>MS2 Minishaker (subsequent model: Ident-Nr.: 0020016017)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile plastic tubes</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>Falcon 352095</td>
			<td>BD Falcon tubes (15 ml, 50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic water bath</td>
			<td>BANDELIN electronic GmbH &#38; Co. KG, Berlin, Germany</td>
			<td style="width:143px;">312</td>
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ultrasonic-augmented primary adult fibroblast isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

The ability of this protocol to isolate adult fibroblasts from solid murine tissue was demonstrated. Viable fibroblasts were obtained that could be used for subsequent experiments such as immunofluorescence staining or proliferation experiments (Figure 2D-F, Figure 5A).
Adult fibroblasts are flat spindle-shaped cells with multiple cellular processes that typically grow in monolayers1...

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Ultrasonic-augmented Primary Adult Fibroblast Isolation

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

ultrasonic-augmented-primary-adult-fibroblast-isolation

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.4K Views.  Technische Universität Dresden. We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Video: Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ventricular stiffness with altered cardiac function. This increase in cardiac ventricular fibrosis is due to activation of resident fibroblasts, although how these cells operate within the 3-dimensional (3-D) heart, at baseline or after activation, is not well understood. To examine how fibroblasts contribute to heart disease and their dynamics in the 3-D heart, a refined CLARITY-based tissue clearing and imaging method was developed that shows fluorescently labeled cardiac fibroblasts within the entire mouse heart. Tissue resident fibroblasts were genetically labeled using Rosa26-loxP-eGFP florescent reporter mice crossed with the cardiac fibroblast expressing Tcf21-MerCreMer knock-in line. This technique was used to observe fibroblast localization dynamics throughout the entire adult left ventricle in healthy mice and in fibrotic mouse models of heart disease. Interestingly, in one injury model, unique patterns of cardiac fibroblasts were observed in the injured mouse heart that followed bands of wrapped fibers in the contractile direction. In ischemic injury models, fibroblast death occurred, followed by repopulation from the infarct border zone. Collectively, this refined cardiac tissue clarifying technique and digitized imaging system allows for 3-D visualization of cardiac fibroblasts in the heart without the limitations of antibody penetration failure or previous issues surrounding lost fluorescence due to tissue processing.

A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy.

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...