Name: isolation of cardiac and vascular smooth muscle cells from adult, juvenile, larval and embryonic zebrafish for electrophysiological studies
Uploaded: 2022-02-09T14:30:00
Description: Watch this Scientific Journal Video about Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies at JoVE.com

This work was supported by NIH grants HL140024 to CGN and HL150277 to CMC. Figure 1 and Figure 2 were created with BioRender.com.

All zebrafish (wild type strain AB, both male and female) were raised, maintained, and handled for the experiments following the guidelines of the Washington University Institutional Animal Care and Use Committee (IACUC).
1. Isolation of atrium, ventricle, and bulbous arteriosus from adult, juvenile, and larval zebrafish
<ol>
	<li>Euthanize fish using cold-shock, i.e., by immersing in 4 &#176;C water, for ~10 s.</li>
	<li>Using curved forceps, transfer fish into a large Petr...

<ol>
	<li>Vascotto, S. G., Beckham, Y., Kelly, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish's+swim+to+fame+as+an+experimental+model+in+biology.">The zebrafish's swim to fame as an experimental model in biology.</a> Biochemistry and Cell Biology. 75 (5), 479-485 (1997).</li><li>Love, D. R., Pichler, F. B., Dodd, A., Copp, B. R., Greenwood, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+for+high-throughput+screens:+The+present+and+future+using+zebrafish.">Technology for high-throughput screens: The present and future using zebrafish.</a> Current Opinion in Biotechnology. 15 (6), 564-571 (2004).</li><li>Ke&#223;ler, M., Rottbauer, W., Just, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+use+of+zebrafish+for+novel+cardiac+drug+discovery.">Recent progress in the use of zebrafish for novel cardiac drug discovery.</a> Expert Opinion on Drug Discovery. 10 (11), 1231-1241 (2015).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. (55), e3165 (2011).</li><li>Brette, 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=Characterization+of+isolated+ventricular+myocytes+from+adult+zebrafish+(Danio+rerio).">Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio).</a> Biochemical and Biophysical Research Communications. 374 (1), 143-146 (2008).</li><li>Sander, V., Sune, G., Jopling, C., Morera, C., Izpisua Belmonte, 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=Isolation+and+in+vitro+culture+of+primary+cardiomyocytes+from+adult+zebrafish+hearts.">Isolation and in vitro culture of primary cardiomyocytes from adult zebrafish hearts.</a> Nature protocol. 8, 800-809 (2013).</li><li>Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+zebrafish+heart+as+a+model+for+human+heart?+An+electrophysiological+study.">Adult zebrafish heart as a model for human heart? An electrophysiological study.</a> Journal of Molecular and Cellular Cardiology. 48 (1), 161-171 (2010).</li><li>Huang, 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=Cardiovascular+consequences+of+KATP+overactivity+in+Cantu+syndrome.">Cardiovascular consequences of KATP overactivity in Cantu syndrome.</a> JCI insight. 3 (15), 137799 (2018).</li><li>Singareddy, S. S., 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=ATP-sensitive+potassium+channels+in+zebrafish+cardiac+and+vascular+smooth+muscle.">ATP-sensitive potassium channels in zebrafish cardiac and vascular smooth muscle.</a> The Journal of Physiology. , (2021).</li><li>Burns, C. G., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hearts+from+zebrafish+embryos.">Purification of hearts from zebrafish embryos.</a> BioTechniques. 40 (3), 274-282 (2006).</li><li>Seiler, C., Abrams, J., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+intestinal+smooth+muscle+development+using+a+novel+sm22&#945;-b+promoter.">Characterization of zebrafish intestinal smooth muscle development using a novel sm22&#945;-b promoter.</a> Developmental Dynamics. 239, 2806-2812 (2010).</li><li>Yang, X. 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=Whole+amount+in+situ+hybridization+and+transgene+via+microinjection+in+zebrafish.">Whole amount in situ hybridization and transgene via microinjection in zebrafish.</a> Shi Yan Sheng Wu Xue Bao. 36 (3), 243-247 (2003).</li><li>Kompella, S. N., Brette, F., Hancox, J. C., Shiels, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenanthrene+impacts+zebrafish+cardiomyocyte+excitability+by+inhibiting+IKr+and+shortening+action+potential+duration.">Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration.</a> The Journal of General Physiology. 153 (2), 202012733 (2021).</li><li>Jou, C. J., Spitzer, K. W., Tristani-Firouzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+effectively+uncouples+the+excitation-contraction+process+in+zebrafish+embryonic+heart.">Blebbistatin effectively uncouples the excitation-contraction process in zebrafish embryonic heart.</a> Cellular Physiology and Biochemistry. 25 (4-5), 419-424 (2010).</li></ol>

Previous methods for isolating zebrafish ventricular myocytes5,6, aimed at generating myocytes for culture or electrophysiological studies, provided cells of lower yield and involved lengthy steps of multiple centrifugations that adversely affected the cell quality and viability. The protocols described here are reliable, cover each of the significant cardiovascular tissues (ventricle, atria, and VSM), and importantly are quite practical for acute isolation of li...

Zebrafish are small teleost fish that have long been used as a model vertebrate organism1 and have recently come to prominence as a viable vertebrate system for high throughput screening of genes and drugs2,3. However, physiological analysis of zebrafish tissues is not well developed. In the cardiovascular system, methods have been described for dissection of zebrafish hearts4 and isolation of ventricular cardiac myocytes5,6,7. There are few detailed descriptions of the effective isolation of atrial myocytes and no reports of vascular smooth muscle (VSM) preparations for patch-clamp studies.
The current work describes methodology for the isolation of zebrafish cardiac and vascular myocytes, viable for electrophysiological and functional studies. This approach includes modifications of previously reported protocols for zebrafish ventricular myocyte isolation5,6 and adapts methods from mammalian VSM cell isolations8, allowing for the isolation of zebrafish vascular smooth muscle cells from the bulbous arteriosus (BA). The protocols result in efficient yields of isolated atrial, ventricular, and VSM cells from zebrafish that can be reliably used in patch-clamp studies for up to 8 h9.
Despite their nearly transparent larvae that develop entirely outside the parental organism, exploring their promised ontogenetic potential in studying cardiovascular development has been limited by challenges in extracting and analyzing tissues at a young&#160;age. The current article addresses this limitation by demonstrating patch-clamp experiments on zebrafish hearts isolated as early as 3 days post-fertilization (dpf), using an adapted, published extraction method10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Centrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td></td>
		</tr>
		<tr>
			<td>19 Guage Needle</td>
			<td>BD</td>
			<td>305187</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione Monoxime (BDM)</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Centrifuge Tubes</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030119479</td>
			<td>For embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier and Digidata 1200 digitizer</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Used for action potential recordings</td>
		</tr>
		<tr>
			<td>Benchtop Mini Centrifuge</td>
			<td>Southern Labware</td>
			<td>MLX-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma-Aldrich</td>
			<td>203390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Strainer Sieve</td>
			<td>Cole-Parmer</td>
			<td>EW-06336-71</td>
			<td>100 &#956;m sieve for embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Collagenase Type H</td>
			<td>Sigma-Aldrich</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>324626</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>Worthington</td>
			<td>LS003118</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Dumont</td>
			<td>Style #5</td>
			<td>Ceramic-coated forceps for adult and juvenile CV tissue isolation (Need two)</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>K2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Petri Dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5981</td>
			<td>For dissociation</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Hematocrit Capillary Tubes</td>
			<td>Kimble Chase</td>
			<td>41A2502</td>
			<td>Soda lime glass for patch pipettes</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003118</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5606</td>
			<td>100 mm x 20 mm, for embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>806552</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>For adult and juvenile zebrafish decapitation</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Fine Forceps</td>
			<td>Dumont</td>
			<td>Style #SF</td>
			<td>For isolating larval CV tissues (Need two)</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>ThermoFisher Scientific</td>
			<td>13687711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanesulfonate (MS222)</td>
			<td></td>
			<td></td>
			<td>For anaesthetizing zebrafish larvae</td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cardiac and vascular smooth muscle cells from adult, juvenile, larval and embryonic zebrafish for electrophysiological studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The above protocols reliably and routinely provide sufficient cardiac and vascular myocytes of consistent quality amenable for patch-clamp studies as recently reported in extensive studies of ATP-sensitive potassium (KATP) channels in wild-type and mutant zebrafish cardiovasculature9. Representative traces of recordings of such KATP channel activity from isolated cardiomyocytes are shown in Figure 3A-C. In the case of cells isola...

Watch this Scientific Journal Video about Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies at JoVE.com

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Studying native ion channel properties using patch clamp electrophysiology requires access to acutely isolated cells. This protocol describes methods to access individual cardiovascular myocytes from zebrafish at different ages. This protocol is robust, simple, easily reproducible, and isolates cardiovascular myocytes from zebrafish in their native state with relatively high yield and viability.To begin, transfer the fish into a large Petri dish partially filled with perfusion buffer and place it under a dissecting microscope. Hold the fish in the non-dominant hand with the ventral side facing the dissection scope. With the fine forceps in the dominant hand, gently tear open the pectoral muscles and fins of the fish to reveal the cardiovascular tissues including atrium, ventricle, and bulbus arteriosus.Gently pull the bulbus arteriosus at the intersecting tip of the bulbus arteriosus and the ventral aorta. Carefully separate the bulbus arteriosus from the aorta by pinching the forceps, locating the atrium's tip into the multiple venous branches that converge. Then, pluck the tip of the atrium off the sinus venosus to isolate the cardiovascular tissues from the rest of the body.To dissect cardiomyocytes, pluck the atrium and ventricle out of the cardiovascular tissue. Make a gentle tear into the ventricle using fine forceps to drain excess blood, which can be ensured when the cardiac tissue turns from bright red to a pale salmon color. Pool the isolated atrium and ventricle into a separate 1.5-milliliter centrifuge tube containing perfusion buffer.Replace the perfusion buffer in the tubes with 750 microliters of digestion buffer. Place the tubes on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute. Allow digestion of the tissues until they become translucent.End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds and replace the supernatant digestion buffer with 750 microliters of stopping buffer. Gently replace the stopping buffer with 500 to 750 microliters of perfusion buffer and triturate the tissues 30 times using a flame-polished Pasteur pipette to disperse the cells. Then, gently triturate the ventricles.For uniform sampling, gently pipette the cells up and down a couple of times to resuspend, before adding a drop of them for corresponding studies. Pluck the adult bulbus arteriosus from the cardiovascular tissues and pool five bulbus arteriosus into a 1.5-milliliter centrifuge tube containing S1 buffer. Replace S1 with 400 microliters of S2 buffer and allow papain digestion of the bulbus arteriosus on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for 20 minutes.Allow the partially digested bulbus arteriosus to settle down for one minute and replace the S2 supernatant with 500 microliters of S3 buffer containing collagenase. Digest the tissues on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for three to five minutes. End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds, and replace the S3 supernatant with 500 microliters of S1.Gently triturate the pelleted tissues to disperse vascular smooth muscle cells.Plate dispersed vascular smooth muscle cells onto glass cover slips of appropriate size. Keep the cover slips at room temperature for 30 minutes for the cells to attach, and use them within the next six hours. Anesthetize the embryos.Then, concentrate the embryos by transferring them to a five-milliliter centrifuge tube, then remove excess media. Wash the embryos with three milliliters of cold perfusion buffer twice, then resuspend them in two milliliters of perfusion buffer. Using the embryonic heart isolation apparatus, draw one milliliter of the embryos into the needle and immediately expel them back into the tube.Pass the fragmented embryos through a 100-micrometer cell strainer sieve placed in a funnel. Collect the filtered hearts in another five-milliliter centrifuge tube. Mix the filtrate well and separate one-milliliter aliquots into 1.5-milliliter centrifuge tubes.Centrifuge all the tubes on a bench-top minicentrifuge for five seconds and discard the supernatant. Carry out serial resuspension of the pellets in the tubes using one milliliter of perfusion buffer. Gently pipette the hearts up and down twice before adding a drop for corresponding studies For cells isolated from bulbus arteriosus, the vascular smooth muscle cells were confirmed by tagln:EGFP expression.Representative traces of recordings of ATP-sensitive potassium channel activity from isolated cardiomyocytes are shown here. Successful single-channel recordings were obtained in patches excised from the embryonic hearts. Ventricular myocytes exhibited stable hyperpolarized membrane potentials, and action potentials were stimulated via current injection through the patch pipette.Atrial myocytes exhibited spontaneous action potential firing. Action potential properties are summarized here. While replacing the buffers during cardiomyocyte dissociation, care should be taken that the digestive tissues are not disturbed.Tissue fragmentation should be accomplished only by pipette trituration. During the collagenous digestion of the bulbus tissues, examine for floating, dilated, and translucent tissues every minute. Once the tissue fragmentation is apparent, cease the digestion.Once the cells are isolated, they can be kept alive for hours and used for electrophysiological analysis using patch clamp techniques. This has allowed us, for instance, to show that ATP-sensitive potassium channels in these zebrafish tissues are essentially identical to those in mammals.

isolation-cardiac-vascular-smooth-muscle-cells-from-adult-juvenile

Research

JoVE Journal

Biology

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...