Name: preparation of acute myocardial tissue slices from biopsies of human neonates and infants with congenital heart disease for physiological measurements
Uploaded: 2017-04-17T16:18:17
Description: In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their ...

The authors acknowledge the technical workshop of the Institute for Neurophysiology for the fabrication of custom-made equipment and the excellent support. We thank Annette K&#246;ster for the skillful technical assistance. This study was supported by the Koeln Fortune programme (T.H., grant no 288/2013) and the B. Braun foundation (T.H. and R.N.).

This study was approved by the local ethics committee of the Medical Faculty of the University of Cologne (reference no. 07-045) and complied with the Word Medical Association Declaration of Helsinki (7th revision, Fortaleza, Brazil, 2013). Written informed consent was given by the parents of each patient.
1. Laboratory Preparation
<ol>
	<li>Switch on the water bath that controls the temperature of a custom-made jacketed vessel (Figure 1). Set the temperature to...

<ol>
	<li>de Boer, T. P., Camelliti, P., Ravens, U., Kohl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+tissue+slices:+organotypic+pseudo-2D+models+for+cardiac+research+&amp;+development.">Myocardial tissue slices: organotypic pseudo-2D models for cardiac research &amp; development.</a> Future Cardiol. 5 (5), 425-430 (2009).</li><li>Wang, 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=Cardiac+tissue+slices:+preparation,+handling,+and+successful+optical+mapping.">Cardiac tissue slices: preparation, handling, and successful optical mapping.</a> Am J Physiol Heart Circ Physiol. 308 (9), H1112-H1125 (2015).</li><li>Yue, L., Feng, J., Li, G. 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A robust model to study ventricular isometric contractions.</a> Cell Physiol Biochem. 20 (6), 837-846 (2007).</li><li>Bussek, A., Schmidt, M., Bauriedl, J., Ravens, U., Wettwer, E., Lohmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+slices+with+prolonged+survival+for+in+vitro+drug+safety+screening.">Cardiac tissue slices with prolonged survival for in vitro drug safety screening.</a> J Pharmacol Toxicol Methods. 66 (2), 145-151 (2012).</li><li>Haustein, 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=Excitation-contraction+coupling+in+zebrafish+ventricular+myocardium+is+regulated+by+trans-sarcolemmal+Ca2++influx+and+sarcoplasmic+reticulum+Ca2++release.">Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.</a> PLoS One. 10 (5), e0125654 (2015).</li><li>Brandenburger, 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=Organotypic+slice+culture+from+human+adult+ventricular+myocardium.">Organotypic slice culture from human adult ventricular myocardium.</a> Cardiovasc Res. 93 (1), 50-99 (2012).</li><li>Piper, H. 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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=The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2,3-butanedione+monoxime.">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am J Physiol Heart Circ Physiol. 302 (1), H262-H269 (2012).</li><li>Farman, G. P., Tachampa, K., Mateja, R., Cazorla, O., Lacampagne, A., de Tombe, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin:+use+as+inhibitor+of+muscle+contraction.">Blebbistatin: use as inhibitor of muscle contraction.</a> Pflugers Arch. 455 (6), 995-1005 (2008).</li></ol>

Cardiac slices bridge the gap between single-cell and complex multicellular models for physiological research1,2. Here a protocol has been introduced describing the preparation of viable cardiac tissue slices from human myocardial explants obtained from neonatal and infantile patients undergoing surgery for congenital heart disease. These slices can be used for studying contractile behavior and electrophysiological properties, among other physiological parameters...

Ex vivo cellular studies on myocardial function rely on a spectrum of models, ranging from isolated single cells to whole-heart preparations such as the Langendorff-perfused heart.
Although isolated cardiomyocytes are the key model for many research questions, they do not entirely reflect the in vivo situation because intercellular interactions and connections to an extracellular matrix (ECM) are missing1,2. Moreover, enzymatic digestion during the dissociation of myocardial tissue can modify the electrophysiological properties of cardiomyocytes. For instance, Yue et al.3 demonstrated that delayed rectifier potassium channel configuration was dependent upon the isolation protocol.
Multicellular preparations, such as the Langendorff-perfused heart or coronary-perfused wedge preparation, on the other hand, provide cardiomyocytes in their native cellular and extracellular environment. This allows for the investigation of phenomena that require interactions such as the development of arrhythmia. To provide proper oxygenation and to cover the metabolic demands, they require arterial perfusion due to their relatively great thickness1,4. This restricts the use of these techniques, especially in human myocardial preparations, as explanted whole hearts or at least large tissue samples with an intact coronary artery are required.
Organotypic tissue slices have been a popular in vitro model for physiological and pathophysiological investigations for many decades. Although well-established for organs such as the brain, liver, and kidney, the use of slice preparations for functional cardiovascular research has gained more interest only recently1,5. With a few exceptions1, myocardial slices have extended the methodological repertoire of cardiovascular research only in the last decade. Many studies have demonstrated that viable cardiac tissue slices of high integrity can be obtained from diverse species, including mouse6,7,8, dog4, guinea pig2,9, rabbit2, zebrafish10, and human4,5,11, and at different developmental stages.
Since the precision vibratome sectioning of slices thinner than 400 &#181;m is feasible, adequate oxygenation and nutrient supply by diffusion can be ensured for cardiac slice preparations1. Cardiac tissue slices show a more in vivo-like profile in terms of cell composition and extracellular matrix than single cardiomyocytes or cell culture models2. As arterial perfusion is not required, this preparation technique can be used for small patient biopsies.
The present protocol describes a method to prepare viable myocardial slices obtained from right ventricular tissue samples. These biopsies are an essential component during surgery on neonate and infant patients with hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF), respectively, and are discarded if not used for experimental purposes.

<table border="0" cellpadding="0" cellspacing="0" width="792">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaCl</td>
			<td style="width:197px;">Carl-Roth</td>
			<td style="width:155px;">HN00.2</td>
			<td style="width:287px;">solution A: 136 mmol/L in aqua dest.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Merck</td>
			<td>1.04936.0500</td>
			<td>solution A: 5.4 mmol/L</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaH2PO4 * 2 H2O</td>
			<td>Carl-Roth</td>
			<td>T879.1</td>
			<td>solution A: 0.33 mmol/L</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgCl2 * 6 H2O</td>
			<td>Merck</td>
			<td>1.05833.0250</td>
			<td>solution A: 1 mmol/L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>Carl-Roth</td>
			<td>HN06.3</td>
			<td>solution A: 10 mmol/L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Carl-Roth</td>
			<td>HN77.3</td>
			<td>solution A: 5 mmol/L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">2,3-Butanedione 
			monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td>solution A: 30 mmol/L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Carl-Roth</td>
			<td>K021.1</td>
			<td>solution A: use to adjust pH to 7.4</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 * 2 H2O</td>
			<td>Merck</td>
			<td>1.02382.0250</td>
			<td>calcium solution: 100 mmol/L in aqua dest.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose Low Melt</td>
			<td>Carl-Roth</td>
			<td>6351.5</td>
			<td>for slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Steel blades</td>
			<td>Campden Instruments</td>
			<td>7550-1-SS</td>
			<td>for slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td>for slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass pasteur pipettes</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>for &#39;slice collector&#39; tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peleus ball</td>
			<td>VWR</td>
			<td>612-2699</td>
			<td>for &#39;Slice collector&#39; tool</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">steel chamber</td>
			<td>custom-made</td>
			<td></td>
			<td style="width:287px;">for embedding tissue into agarose; 
			cylindric shape (inner: &#216;: 1.5 cm, depth 0.9 cm; outer: &#216;: 2.0 cm, depth 1.1 cm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">instant adhesive</td>
			<td>Henkel AG</td>
			<td>PSG2C</td>
			<td>Pattex Ultra Gel; for slicing</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Jacketed vessel</td>
			<td>custom-made</td>
			<td></td>
			<td style="width:287px;">to maintain temperature of agarose in beaker at 37&#176;C; made of steel with a plexiglass bottom</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Grant</td>
			<td>Sub 14</td>
			<td>for temperature control of jacketed vessel</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Water pump</td>
			<td>Aquarium 
			Systems</td>
			<td>Mini Jet MN404</td>
			<td>for temperature control of jacketed vessel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td>31980022</td>
			<td>for force measurements</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose</td>
			<td>Life Technologies</td>
			<td>61965026</td>
			<td>for microelectrode recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E4031 dihydrochloride</td>
			<td>Abcam</td>
			<td>ab120158</td>
			<td>for microelectrode recordings</td>
		</tr>
	</tbody>
</table>

preparation of acute myocardial tissue slices from biopsies of human neonates and infants with congenital heart disease for physiological measurements

In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their complexity, ranging from isolated cardiomyocytes to multicellular 3-dimensional tissue preparations, such as the Langendorff-perfused heart or coronary-perfused wedges. Cardiac tissue slices bridge the gap between these models, as their relatively low thickness overcomes the need for arterial perfusion, while the native cellular alignment and extracellular matrix structure are preserved. This enables the use of tissue when coronary perfusion is not available (e.g., tissue excised during surgery for congenital heart disease). The present protocol describes the preparation of viable cardiac slices from myocardial explants from neonate and infant patients undergoing surgery for congenital heart disease. Upon extraction, the myocardial tissue is transferred to oxygenated, ice-cold, low-calcium solution and transported to the laboratory. Thereafter, the tissue is pre-cut, embedded into low-melting agarose, and sectioned with a vibratome. Tissue recovery is promoted by the stepwise increase of calcium concentration, followed by gradual rewarming to 37 &#176;C for 1 h in the measurement solution. Afterwards, the obtained acute myocardial slices can be used for physiological experiments. Representative results for isometric force measurements and action potential recordings are provided. The importance of the solution and the vibratome parameters to the preparation of viable cardiac slices, as well as limitations regarding the control of the fiber alignment and long-term culture, are discussed.

Pictures of typical myocardial tissue slices obtained using the present protocol are shown in Figure 2. The prepared slices can be used for physiological measurements, such as force measurements or electrophysiological recordings.
For force measurements, the tissue slices were mounted onto J-shaped steel needles connected to an isometric force transducer. The slices were immersed in the measurement ...

Cardiac slices are a unique model for cardiovascular research and bridge the gap between single-cell and whole-heart models. This protocol describes the preparation of viable cardiac slices from myocardial tissue samples excised during surgery for congenital heart disease.

Preparation of Acute Myocardial Tissue Slices from Biopsies of Human Neonates and Infants with Congenital Heart Disease for Physiological Measurements

In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their ...

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