Name: isolation of human ventricular cardiomyocytes from vibratome-cut myocardial slices
Uploaded: 2020-05-10T13:30:00
Description: Watch this Scientific Journal Video about Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices at JoVE.com

We would like to thank Andreas Dendorfer from the Walter-Brendel-Centre of Experimental Medicine, LMU Munich, for help with the slicing protocol. For providing human myocardial tissue samples we would like to thank Ghazali Minabari and Christian Heim from the Department of Cardiac Surgery, University Hospital Erlangen, Hendrik Milting from the Erich &#38; Hanna Klessmann Institute, Ruhr-University Bochum and Muhannad Alkassar from the Department of Pediatric Cardiology, University Hospital Erlangen. For support with flow cytometry we would like to thank Simon V&#246;lkl and colleagues from the translational research center (TRC), University Hospital Erlangen. We would also like to thank Lorenz McCargo and Celine Gr&#252;ninger from the Institute of Cellular and Molecular Physiology Erlangen for excellent technical support.
This work was supported by the DZHK (German Centre for Cardiovascular Research), by the Interdisciplinary Centre for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-N&#252;rnberg, and the Universit&#228;tsbund Erlangen-N&#252;rnberg.

All experiments with rats were approved by the Animal Care and Use Committee Mittelfranken, Bavaria, Germany. Collection and use of human cardiac tissue samples was approved by the Institutional Review Boards of the University of Erlangen-N&#252;rnberg and the Ruhr-University Bochum. Studies were conducted according to Declaration of Helsinki guidelines. Patients gave their written informed consent prior to tissue collection.
Female Wistar rats (150&#8211;200 g) were commercially obtained, ane...

Although the isolation of living cardiomyocytes was established more than 40 years ago and is still a prerequisite for many experimental approaches in cardiac research, it remains a difficult technique with unpredictable outcomes. Cardiomyocyte isolation via perfusion of the coronary arteries with enzyme solution is commonly used for hearts of small animals and yields large numbers of viable cells. However, this requires a relatively complex system and expertise. Furthermore, most human tissue samples are not suited for ...

A seminal technique that has paved the way to important insights into cardiomyocyte physiology is the isolation of living ventricular cardiomyocytes from intact hearts1. Isolated cardiomyocytes can be used to study normal cellular structure and function, or the consequences of in vivo experiments; for example, to assess changes in cellular electrophysiology or excitation-contraction coupling in animal models of cardiac disease. Additionally, isolated cardiomyocytes can be used for cell culture, pharmacological interventions, gene transfer, tissue engineering, and many other applications. Therefore, efficient methods for cardiomyocyte isolation are of fundamental value to basic and translational cardiac research.
Cardiomyocytes from small mammals, such as rodents, and from larger mammals, such as pigs or dogs, are commonly isolated by coronary perfusion of the heart with solutions containing crude collagenases and/or proteases. This has been described as the &#8220;gold standard&#8221; method for cardiomyocyte isolation, resulting in yields of up to 70% of viable cells2. The approach has also been used with human hearts, resulting in acceptable cardiomyocyte yields3,4,5. However, because coronary perfusion is only feasible if the intact heart or a large myocardial wedge containing a coronary artery branch is available, most human cardiac specimens are not suited for this approach due to their small size and a lack of appropriate vasculature. Therefore, the isolation of human cardiomyocytes is challenging.
Human myocardial specimens mostly consist of tissue chunks of variable size (approximately 0.5 x 0.5 x 0.5 cm to 2 x 2 x 2 cm), obtained through endomyocardial biopsies6, septal myectomies7, VAD implantations8, or from explanted hearts9. The most common procedures for cardiomyocyte isolation start with mincing the tissue using scissors or a scalpel. Cell-to-cell contacts are then disrupted by immersion in calcium-free or low-calcium buffers. This is followed by multiple digestion steps with crude enzyme extracts or purified enzymes like proteases (e.g., trypsin), collagenase, hyaluronidase, or elastase, resulting in a disintegration of the extracellular matrix and liberation of cardiomyocytes. In a final, critical step, a physiological calcium concentration has to be carefully restored, or cellular damage can occur due to the calcium-paradox10,11,12. This isolation approach is convenient but usually inefficient. For instance, one study found that nearly 1 g of myocardial tissue was required to obtain a sufficient number of cardiomyocytes suitable for subsequent experiments13. A possible reason for low yields is the relatively harsh method of mincing the tissue. This may particularly damage cardiomyocytes located at the chunk edges&#160;although these myocytes are most likely to be released by enzymatic digestion.
Another aspect that may influence isolation efficiency and quality of cells obtained from human specimens is the duration of tissue ischemia. Most protocols mention short transportation times to the laboratory as a prerequisite for good&#160;results. This restricts the study of human ventricular cardiomyocytes to laboratories with nearby cardiac surgery facilities. Together, these restrictions hamper the verification of important findings from animal models in human cardiomyocytes. Improved isolation protocols that allow for high cardiomyocyte yields from small amounts of tissue, preferably without serious damage after extended transportation times, are therefore desirable.
Described here is an isolation protocol based on the enzymatic digestion of thin myocardial tissue slices generated with a vibratome14,15. We demonstrate that isolation from tissue slices is much more efficient than that from tissue chunks minced with scissors. The described method not only allows for high yields of viable human cardiomyocytes from small amounts of myocardial tissue but is also applicable to specimens stored or transported in cold cardioplegic solution for up to 36 h.

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-butanedionemonoxime</td>
			<td>Carl Roth</td>
			<td>3494.1</td>
			<td>Purity&#62;99%</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Carl Roth</td>
			<td>163.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth</td>
			<td>5239.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine monohydrate</td>
			<td>Alfa Aesar</td>
			<td>B250009</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth</td>
			<td>9105.3</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>P017.1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Carl Roth</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>Fluka Biochemica</td>
			<td>49450</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting-point agarose</td>
			<td>Carl Roth</td>
			<td>6351.5</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Carl Roth</td>
			<td>A537.1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich</td>
			<td>M-7506</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Carl Roth</td>
			<td>8551.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma Aldrich</td>
			<td>T8691</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Di-8-ANEPPS</td>
			<td>Thermo Fisher Scientific</td>
			<td>D3167</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase CLS type I</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td>Used for human tissue at 4 mg/mL 
			(activity: 280 U/mg)</td>
		</tr>
		<tr>
			<td>Collagenase CLS type II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td>Used for rat tissue at 1.5 mg/mL 
			(activity 330 U/mg)</td>
		</tr>
		<tr>
			<td>Protease XIV</td>
			<td>Sigma Aldrich</td>
			<td>P8038</td>
			<td>Used for rat tissue at 0.5 mg/mL 
			(activity &#8805; 3.5 U/mg)</td>
		</tr>
		<tr>
			<td>Proteinase XXIV</td>
			<td>Sigma Aldrich</td>
			<td>P5147</td>
			<td>Used for human tissue at 0.5 mg/mL 
			(activity: 7-14 U/mg)</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyzer (LSR Fortessa)</td>
			<td>BD Bioscience</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 15 mL</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube, 50 mL</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact shaker</td>
			<td>Edmund B&#252;hler</td>
			<td>KS-15 B control</td>
			<td>Agitation direction: horizontal</td>
		</tr>
		<tr>
			<td>Disposable plastic pasteur-pipettes</td>
			<td>Carl Roth</td>
			<td>EA65.1</td>
			<td>For cell trituration use only pipettes with an inner tip diameter &#8805;2 mm</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Heatblock</td>
			<td>VWR</td>
			<td>BARN88880030</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon net filter, 180 &#181;m</td>
			<td>Merck</td>
			<td>NY8H04700</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Dish 100, Standard</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Dish 35, Standard</td>
			<td>Sarstedt</td>
			<td>83.3900</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Dish 60, Standard</td>
			<td>Sarstedt</td>
			<td>83.3901</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome (VT1200S)</td>
			<td>Leica</td>
			<td>1491200S001</td>
			<td>Includes VibroCheck for infrared-assisted correction of z-deflection</td>
		</tr>
	</tbody>
</table>

isolation of human ventricular cardiomyocytes from vibratome-cut myocardial slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

To verify isolation efficiency, the protocol was used with rat myocardium and the resulting number of viable myocytes was compared with the numbers obtained by isolation via coronary perfusion and by isolation from small tissue chunks (chunk isolation, Figure 2). Chunk isolation and isolation from tissue slices were performed from the same hearts. For the isolation via coronary perfusion, however, the whole heart was used. Coronary perfusion yielded predominantly rod-shaped and cross-striate...

Watch this Scientific Journal Video about Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices at JoVE.com

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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