Name: preparation of human myocardial tissue for long-term cultivation
Uploaded: 2022-06-02T14:10:00
Description: Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Research was funded by DZHK grants 81Z0600207 (JH, PS, and DM) and 81X2600253 (AD and TS).
The authors would like to thank Claudia Fahney, Mei-Ping Wu, and Matthias Semisch for their support in preparing the set-ups, as well as for the regular maintenance of the tissue cultivation.

Tissue collection for the experiments described here was approved by the Institutional Review Boards of the University of Munich and the Ruhr-University Bochum. Studies were conducted according to Declaration of Helsinki guidelines. Patients gave their written informed consent prior to tissue collection.
1. Tissue acquisition
<ol>
	<li>Obtain human tissue from patients undergoing heart transplantation or cardiac surgery.</li>
	<li>Before procuring the tissue, prepare 2 L of ...

<ol>
	<li>George, S. A., Brennan, J. A., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+cardiac+electrophysiology+assessment+by+dual+voltage+and+calcium+optical+mapping+of+human+organotypic+cardiac+slices.">Preclinical cardiac electrophysiology assessment by dual voltage and calcium optical mapping of human organotypic cardiac slices.</a> Journal of Visualized Expereiments: JoVE. (160), e60781 (2020).</li><li>Lu, 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=Progressive+stretch+enhances+growth+and+maturation+of+3D+stem-cell-derived+myocardium.">Progressive stretch enhances growth and maturation of 3D stem-cell-derived myocardium.</a> Theranostics. 11 (13), 6138-6153 (2021).</li><li>Pontes Soares, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+and+3D-organized+cardiac+cells+shows+differences+in+cellular+morphology,+adhesion+junctions,+presence+of+myofibrils+and+protein+expression.">2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.</a> PloS one. 7 (5), 38147 (2012).</li><li>Klumm, M. J., 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=Long-term+cultivation+of+human+atrial+myocardium.">Long-term cultivation of human atrial myocardium.</a> Frontiers in Physiology. 13, 839139 (2022).</li><li>Krane, 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=Sequential+defects+in+cardiac+lineage+commitment+and+maturation+cause+hypoplastic+left+heart+syndrome.">Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome.</a> Circulation. 144 (17), 1409-1428 (2021).</li><li>Miller, J. 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=Heart+slice+culture+system+reliably+demonstrates+clinical+drug-related+cardiotoxicity.">Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.</a> Toxicology and Applied Pharmacology. , 406 (2020).</li><li>Fischer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Kang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organotypic+cultured+cardiac+slices:+new+platform+for+high+throughput+preclinical+human+trials.">Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials.</a> Scientific Reports. 6 (1), 1-13 (2016).</li><li>Ou, Q., 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=Slicing+and+culturing+pig+hearts+under+physiological+conditions.">Slicing and culturing pig hearts under physiological conditions.</a> Journal of Visualized Experiments: JoVE. (157), e60913 (2020).</li><li>Ou, Q., 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=Physiological+biomimetic+culture+system+for+pig+and+human+heart+slices.">Physiological biomimetic culture system for pig and human heart slices.</a> Circulation research. 125 (6), 628-642 (2019).</li><li>Watson, S. 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=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 1-15 (2019).</li><li>Abu-Khousa, 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=The+degree+of+t-system+remodeling+predicts+negative+force-frequency+relationship+and+prolonged+relaxation+time+in+failing+human+myocardium.">The degree of t-system remodeling predicts negative force-frequency relationship and prolonged relaxation time in failing human myocardium.</a> Frontiers in Physiology. 11, 182 (2020).</li><li>Bojkova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+infects+and+induces+cytotoxic+effects+in+human+cardiomyocytes.">SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.</a> Cardiovascular Research. 116 (14), 2207-2215 (2020).</li><li>Esfandyari, D. A. -. 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=MicroRNA-365+regulates+human+cardiac+action+potential+duration.">MicroRNA-365 regulates human cardiac action potential duration.</a> Nature Communications. 13 (1), 1-15 (2022).</li><li>Moretti, 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=Somatic+gene+editing+ameliorates+skeletal+and+cardiac+muscle+failure+in+pig+and+human+models+of+Duchenne+muscular+dystrophy.">Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.</a> Nature Medicine. 26 (2), 207-214 (2020).</li><li>Pitoulis, F. G., 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=Remodelling+of+adult+cardiac+tissue+subjected+to+physiological+and+pathological+mechanical+load+in+vitro.">Remodelling of adult cardiac tissue subjected to physiological and pathological mechanical load in vitro.</a> Cardiovascular Research. 118 (3), 814-827 (2021).</li><li>Curtis, T. M., Scholfield, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nifedipine+blocks+Ca2++store+refilling+through+a+pathway+not+involving+L-type+Ca2++channels+in+rabbit+arteriolar+smooth+muscle.">Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.</a> The Journal of Physiology. 532 (3), 609-623 (2001).</li><li>de Weille, J. R., Schweitz, H., Maes, P., Tartar, A., Lazdunski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calciseptine,+a+peptide+isolated+from+black+mamba+venom,+is+a+specific+blocker+of+the+L-type+calcium+channel.">Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.</a> Proceedings of the National Academy of Sciences. 88 (6), 2437-2440 (1991).</li><li>Schleifer, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+molecular+modelling+study+of+the+calcium+channel+blockers+nifedipine+and+black+mamba+toxin+FS2.">Comparative molecular modelling study of the calcium channel blockers nifedipine and black mamba toxin FS2.</a> Journal of Computer-Aided Molecular Design. 11 (5), 491-501 (1997).</li><li>Thomas, G., Chung, M., Cohen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dihydropyridine+(Bay-K8644)+that+enhances+calcium+currents+in+guinea+pig+and+calf+myocardial+cells.+A+new+type+of+positive+inotropic+agent.">A dihydropyridine (Bay-K8644) that enhances calcium currents in guinea pig and calf myocardial cells. A new type of positive inotropic agent.</a> Circulation Research. 56 (1), 87-96 (1985).</li><li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+slices+come+to+age:+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research.">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li><li>Watson, S. 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=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li></ol>

In the past, cardiovascular research has made great advances in the cultivation of cardiomyocytes. However, the 3D cultivation of cardiomyocytes with intact geometry is not yet well-established. Compared to previous protocols applied for ex vivo cultivation of myocardial tissue, the protocol that we described here resembles the in vivo environment of the tissue more closely. Moreover, the application of pre- and afterload allows for a more biomimetic environment. We are able to fully analyze and underst...

In the past decade, in vitro cultivation of myocardial cells has made great advances, ranging from 2D and three-dimensional (3D) techniques to the use of organoids and induced pluripotent stem cells differentiated into cardiac myocytes1,2,3. Ex vivo and primary cell cultivations have shown to be of great value, especially for genetic studies and drug development4,5,6. Using human tissues improves the translational value of the results. Long-term 3D cultivation of myocardial tissues with intact geometry, however, is not well-established. The intact geometry is a key feature to mimic in vivo conditions, as proper cardiac function, communication between different cells, as well as cell-matrix interactions are prerequisites. Myocardial tissue cultivation went through various phases of development. The success rate and stability of ex vivo myocardial tissue cultivation were initially quite low, but recent approaches have yielded promising results7,8,9,10,11.
Among those, Fischer et al. were the first to demonstrate that viability and contractile performance of human myocardial tissue can be maintained in ex vivo cell cultivation for many weeks7. Their technique was based on thin tissue slices cut from explanted human myocardium, which were mounted in newly developed cultivation chambers that provided defined biomechanical conditions and continuous electrical stimulation. This cultivation method closely resembles the in vivo function of myocardial tissue, and has been reproduced by several independent research groups2,12,13,14,15. Importantly, the chambers used by Fischer et al. also enabled continuous registration of developed forces for up to 4 months, and thus opened unprecedented opportunities for physiological and pharmacological research on intact human myocardium7.
Similar techniques were independently developed by other groups and applied to human, rat, porcine, and rabbit myocardium7,10,11. Pitoulis et al. subsequently developed a more physiological method, which reproduces the normal force-length relation during a contraction cycle, but is less suitable for high-throughput analysis16. As such, the general approach of biomimetic cultivation can be regarded as a further step into the reduction, refinement, and replacement (3R) of animal experiments.
However, exploitation of this potential requires standardized procedures, high content analyses, and a high throughput level. We present a technique that combines automated slicing of living human myocardium with in vitro maintenance in a biomimetic cultivation system that has become commercially available (see Table of Materials). With the proposed approach, the number of individual slices that can be generated from a single transmural myocardial specimen is only limited by the processing time. A specimen of sufficient size and quality (3 cm x 3 cm) often yields 20-40 tissue slices being conveniently cut with an automated vibratome. These slices can be placed in cultivation chambers belonging to the system. The chambers allow for electrical stimulation, the parameters of which can be modulated (i.e., pulse duration, polarity, rate, and current), as well as the adjustment of pre- and afterload, using spring wires inside the chambers. The contraction of each slice is registered from the movement of a small magnet attached to a spring wire and displayed as an interpretable graph. Data can be recorded at all times and analyzed using freely available software. Aside from the constant baseline pacing, scheduled protocols can be performed to functionally assess their refractory period, stimulation threshold, post-pause-potentiation, and force-frequency relation.
This long-term biomimetic cultivation of multiple myocardial slices from an individual heart paves the way for future ex vivo research in both human and animal tissue, and facilitates the screening for therapeutic and cardiotoxic drug effects in cardiovascular medicine. It has already been applied to various experimental approaches2,12,13,15. Here, we give a detailed step-by-step description of the preparation of human tissue and provide solutions for frequently encountered cultivation problems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low melting point</td>
			<td>Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bay-K8644</td>
			<td>Cayman Chemical</td>
			<td>19988</td>
			<td></td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime)</td>
			<td>Sigma</td>
			<td>B0753-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*H2O</td>
			<td>Merck</td>
			<td>2382.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Calciseptine</td>
			<td>Alomone Labs</td>
			<td>SPC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose*H2O</td>
			<td>AppliChem</td>
			<td>A3730.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>BBraun</td>
			<td>3703452</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl</td>
			<td>BBraun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>SAV LP GmbH</td>
			<td>UN1219</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-X-supplement</td>
			<td>Gibco</td>
			<td>5150056</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04933.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>AppliChem</td>
			<td>A1036.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4*H2O</td>
			<td>Merck</td>
			<td>1.06346.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin x100</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cabinet</td>
			<td>Thermo Scientific</td>
			<td>KS15</td>
			<td></td>
		</tr>
		<tr>
			<td>Frigomix waterpump and cooling + BBraun Thermomix BM</td>
			<td>BBraun</td>
			<td></td>
			<td>In-house made combination of cooling and heating solution.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Binder</td>
			<td>CB240</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoDish bioreactor system</td>
			<td>InVitroSys GmbH</td>
			<td>MyoDish 1</td>
			<td>Myodish cultute system</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200s</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 37 degrees</td>
			<td>Haake</td>
			<td>SWB25</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath 80 degrees</td>
			<td>Daglef Patz KG</td>
			<td>7070</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL plastic single-use beaker</td>
			<td>Sarstedt</td>
			<td>75.562.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration unit, Steritop Quick Release</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 0.9 x 70 mm 20G</td>
			<td>BBraun</td>
			<td>4665791</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic triangles</td>
			<td></td>
			<td></td>
			<td>In-house made</td>
		</tr>
		<tr>
			<td>Razor Derby premium</td>
			<td>Derby Tokai</td>
			<td>B072HJCFK6</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Gillette Silver Blue</td>
			<td>Gillette</td>
			<td>7393560010170</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel disposable</td>
			<td>Feather</td>
			<td>02.001.30.020</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10 mL Luer tip BD Discardit</td>
			<td>BBraun</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 10 cm</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 3.5 cm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of human myocardial tissue for long-term cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

The contraction of the myocardial slices was displayed on the computer screen after insertion of the cultivation chamber into its corresponding connector (Figure 3). Contraction of the human myocardial slices started immediately upon stimulation. The slices hypercontracted for 5-10 min. This was visible as an increase of diastolic forces, caused by a tonic contracture of damaged tissue fractions. This process was reverted to varying degrees within 1-1.5 h. After stabilizing, human LV tissue ...

Watch this Scientific Journal Video about Preparation of Human Myocardial Tissue for Long-Term Cultivation at JoVE.com

Preparation of Human Myocardial Tissue for Long-Term Cultivation

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Using this protocol, human left ventricular tissue slices can be cultivated ex vivo in a biomimetic chamber. The application of pre and afterloads improves the resemblance of the physiological environment. This technique allows for continuous registration of tissue contraction.User-defined stimulation protocols allow for the assessment of vital contraction parameters like post-pause potentiation, stimulation threshold, force-frequency relation, and refractory period. The long-term cultivation of myocardial slices, using this setup, will pave the way for future ex vivo research, facilitating this screening for therapeutic and cardiotoxic drug effects in cardiovascular medicine. To begin, submerge the cultivation chambers and graphite electrodes in one liter of a 10%isopropanol solution and agitate overnight.The following day, transfer the chambers to a 100%isopropanol solution for 3 minutes, then allow the chambers and graphite electrodes to air dry under a laminar flow hood. Attach a circuit board to each chamber according to the available positions on the rocker. Place two graphite electrodes on the circuit board as per the manufacturer's instructions, then place a 35-millimeter Petri dish lid on the chamber to prevent infection.Next, fill the cutting tray up to 90 to 95%with slicing buffer. Transfer the tissue sample to a 100-millimeter Petri dish filled with cold slicing buffer, and place the dish on a cooling plate at 4 degrees Celsius. Remove endocardial trabeculae by holding the endocardium with tweezers, then use scissors to cut away approximately 3 millimeters of endocardial tissue.Fixate the cut tissue sample, endocardial side up to a 2-by-2-centimeter rubber patch using four 0.9-by-70-millimeter 20-gauge needles that are fixed in a square position. Ensure that the diagonal edge of each needle tip is pointing inward, enhancing fixation and preventing damage to the myocardium. Using a scalpel, cut away all the excess tissue outside the four needle squares.Using tweezers, place the trimmed sample on a sterile piece of tissue for 10 seconds to remove excess slicing buffer. Next, remove the agarose syringe from the water bath and submerge the sample in agarose. Let the agarose solidify for five minutes on a cooling plate.The endocardial side of the sample must be visible in the agarose. Using tweezers, place the epicardial side of the sample contained in agarose on top of the glued area, then gently press the agarose-containing sample from the top with a blunt tool without cutting or damaging the agarose. Let the glue solidify for 1 minute.Set the vibration amplitude to 1 millimeter, initial cutting speed to 0.07 millimeter per second, and thickness of the slice to 300 microns. After connecting the cultivation system to a computer, start the corresponding software program. Set the rocker speed to 60 rpm and preset the stimulation parameters.For human cardiac slices, set the standard stimulation to biphasic impulses with 50 milliamp or current consisting of 3 milliseconds positive current, 1 millisecond pause, and a 3-millisecond pulse of inverted current at a pacing rate of 30 beats per minute or bpm, then check the electrode indicators of the software and verify that the electrodes of the cultivation chambers are working correctly. Using tweezers, separate the agarose from the tissue. Avoid touching the tissue and handle it carefully as any damage to the tissue will reduce the success rate of the cultivation.To attach two plastic triangles to a sample, place 1 microliter of glue on a sterile Petri dish lid. Use a hooked tweezer to pick up one autoclaved plastic triangle. Quickly dip the front edge of the triangle into the glue and paste the triangle onto the sample perpendicular to the cardiomyocyte alignment.Repeat for the other triangle. Using a scalpel, trim off tissue exceeding the triangle width and place the slice with the two mounted triangles back into the cutting tray containing slicing buffer. Remove the medium-filled cultivation chamber from the incubator.Select one prepared slice and insert it into the chamber by connecting one triangle to each pin. Adjust the distance between the mounting pins to the sample size. Ensure that the sample is submerged in the medium.After placing the dish onto the rocker, decrease the preload by turning the adjusting screw counterclockwise until the baseline of the corresponding graph on the computer screen does not change anymore, then carefully increase the preload or tension by turning the adjusting screw clockwise. For chambers with high stiffness, continue until the corresponding baseline in the graph has increased by 1, 000 to 1, 200 units corresponding to one millinewton preload. After preparing the fresh cultivation medium, pre-warm the medium in a water bath or hot air incubator at 37 degrees Celsius for 30 to 45 minutes.Remove medium from the chamber, leaving approximately 0.8 milliliters in the chamber, then add 1.6 milliliters of fresh medium to the same chamber, making a total volume of medium to 2.4 milliliters per chamber. Finally, place the cover of the chamber back and place the cultivation chamber into its respective position. The readout of the contraction of five myocardial slices during a typical stimulation consisted of four distinct sections of post-pause potentiation, stimulation threshold, force-frequency relation, and refractory period.Compared to control, the contraction force of the slices treated with calcium antagonists, nifedipine and calciseptine, was decreased within 10 minutes. In contrast, the voltage-gated calcium channel agonist, Bay-K8644, increased the contraction force. The post-pause potentiation of the control and treated slices was assessed for the intracellular calcium release from the sarcoplasmic reticulum.The control slice did not show any changes. In the presence of calciseptine and nifedipine, the inhibition of the L-type calcium channels led to potentiation of the first contraction after a pause of 50 seconds, reflecting a higher relative contribution of intracellular calcium release to total contractility. The opposite effect was observed with Bay-K8644, which stimulates the entry of extracellular calcium via the L-type calcium channels.In force-frequency relation analysis, no change was observed in the control slice. The calciseptine treatment did not change the ability of the slice to follow the stimuli upon an increase of the stimulation frequency when comparing the pre and post-treatment data. Compared to calciseptine, nifedipine prevented an increase in contractility at higher pacing rates and reduced the maximum capture rate at 80 bpm.With Bay-K8644, increased contraction force at very low stimulation frequencies was observed. However, at frequencies higher than 50 bpm, the contraction force was lower than during the pre-treatment condition. Excised tissue should be quickly transferred to 4 degrees cardioplegia.After slicing, plastic triangles must be attached perpendicular to the fiber direction. The preload should not exceed 1500 millinewton. Biomimetic slice culture can help researchers to utilize genetic manipulations as well as cell-based therapies for studying regeneration and repair in adult human myocardium.

Research

JoVE Journal

Medicine

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...