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 ...

This protocol works with samples from human and animal hearts, even after hours of transportation time. Up to 200 myocytes per milligram can be isolated from vibratome-cut tissue slices. Electrophysiological, structural and biochemical analysis of cardiomyocytes, helps to discover and better understand, mechanisms of cardiac disease.After filling a 100 millimeter tissue culture dish, with 20 milliliters of cold cutting solution, place the tissue sample in the culture dish. Keep the culture dish on a plate cooled to 4 degrees Celsius. Keep in mind that living tissue samples from animals and especially humans are potentially infectious.Use scissors or scalpel, to remove excess fibrotic tissue and epicardial fat, from the sample. For optimal vibratome processing of larger tissue samples, use the scalpel to cut eight millimeter cubes from the tissue. For smaller samples from biopsies, this is not necessary.One can identify the epicardium by the epicardial fat layer. After removing excess fat, place the tissue block, into the dish, with the epicardium facing down. To prepare for tissue embedding, boil 400 milligrams of low melting point agarose in ten milliliters of cutting solution.Fill a ten milliliter syringe with the hot dissolved agarose gel. Seal the syringe and place it in a 37 degrees Celsius water bath for at least 15 minutes, to allow the agarose to equilibrate. Using forceps, move the trim specimen into a clean 35 millimeter tissue culture dish, with the epicardium facing downward.Then, remove excess fluid from the specimen, with a sterile swab. Secure the specimen against movement, by holding it with forceps. And empty the syringe of agarose over top of it, making sure to completely immerse the specimen.Immediately placed the dish on ice and let the agarose solidify for 10 minutes. Mount an unused razor blade to the blade holder of the vibratome. If possible, calibrate the vibratome, by adjusting the Z deflection of the blade.Use a scalpel to excise an agarose tissue block that fits the specimen holder of the vibratome. To ensure stability, make sure the tissue is still sufficiently immersed in agarose. With agarose margins, of at least eight millimeters.Apply a thin layer of cyanoacrylate glue to the specimen holder. And gently press the agarose tissue block, onto the holder. Build a vibratome bath, with cutting solution.Then, fill the outer cooling tank of the vibratome with crushed ice, to maintain a temperature of four to six degrees Celsius, throughout the cutting process. Place the specimen holder, containing the agarose tissue block, into the vibratome bath. Using the vibratome settings described in the manuscript, generate 300 microliter thick slices.It is crucial to create sections in parallel epicardium because this is how cardiomyocyte fibers are aligned in the ventricular wall. Otherwise, there will be too much damage. Use a standard light microscope, to check the alignment of the cardiomyocytes and the tissue slices.To avoid damaging the tissue, hold the agarose, instead of the tissue itself. A cardiomyocyte fibers, should be uniformly aligned and the myocytes, should not be contracted. Place a heat plate on the lab shaker and warm it to 37 degrees Celsius.Start the lab shaker at 65 RPM. Dissolve the proteinase in two milliliters of solution one. And dissolve the collagenase, in two milliliters of solution one.But do not mix the proteinase and the collagenase. Add calcium chloride to the collagenase solution, for a final concentration, of five micromolar. Incubate both solutions at 37 degrees Celsius.Use forceps to transfer a tissue slice to a clean 60 millimeter tissue culture dish. With five millimeters of pre-chilled cutting solution. Keeping the dish on ice or a cold plate, carefully remove the agarose from the tissue, using a blade or forceps.To perform the initial wash of the tissue slices, place a clean 35 millimeter tissue culture dish on the heat blade. And fill it, with two millimeters of pre-warmed solution one. Transfer one or two tissue slices to the prepared dish.Then use a one millimeter pipette, for the washing steps. After washing the slices, remove solution one from the dish and add two milliliters of the proteinase solution. Incubate the slices for 12 minutes on the heat blade, with the shaker at 65 RPM.Wash the tissue slices twice more, with two milliliters of pre-warmed solution one. Next, remove solution one from the dish and add two milliliters of collagenase solution. Incubate the slices for at least 30 minutes on the heat blade, with shaking at 65 RPM.After 15 minutes of incubation, check for free individual myocytes, by examining the dish under a light microscope. Continue checking every five minutes, until individual myocytes are visible. Then, alt digestion by washing the slices twice, with two milliliters of pre-warmed solution two.And refill the dish, with two milliliters of solution two. Carefully pull the fibers of the tissue slice apart, using fine forceps. Continue to work carefully, to avoid damaging the cardiomyocytes.And pipette several times, with a single use past your pipette. Then, confirm that the rod-shaped cardiomyocytes have separated, by examining the dish, under a light microscope. Place the dish back on the heat plate at 37 degrees Celsius and agitate by shaking.By adding 10 millimolar and 100 millimolar calcium chloride stock solutions, slowly increase the calcium concentration of the tissue suspension from five micromolar to 1.5 millimolar. When the calcium increase has complete, use forceps to remove any remaining undigested tissue chunks. First, stop the agitation of the suspension.Next, using a 1000 microliter pipette, slowly remove 1/3 of the solution, from the top of the suspension. Avoid aspiration of the cardiomyocytes. Then add 700 microliters of solution three, to the cells.After 10 minutes of agitation, repeat these wash steps. Transfer the suspension to a 15 milliliter centrifuge tube. And allow the cardiomyocytes to sediment at room temperature, for a minimum of 10 minutes and a maximum of 30 minutes.Remove the supernatant completely. And resuspend the pellet in modified tyrode solution or the desired experimentation buffer. Using a light microscope, verify the cell quality.30 to 50%of the cardiomyocytes should be rod shaped, smooth without membrane blebs. And display, clear cross striations. To verify isolation efficiency, the protocol was applied to rat myocardium.And compared with isolation via coronary perfusion and isolation, from small tissue chunks. A lower proportion of rod shaped cells was observed when using the protocol, then after isolation by perfusion. But the total number was still high.In contrast, isolation from tissue chunks, yielded fewer rod-shaped cells. Flow cytometry, was used to compare the results quantitatively. The isolation of cardiomyocytes from tissue slices, does not reach to the yield obtained by coronary perfusion.But provides, significantly higher yields than myocyte isolation, from tissue chunks. Next, the protocol was applied to human myocardium. Isolation from myocardial slices, yielded Hyde numbers and a large proportion of rod shaped human myocytes.And only a small proportion of, rounded cardiomyocytes. Isolation from myocardial slices, resulted in a strikingly higher number of rod-shaped myocytes, than isolation from tissue chunks. Photometric quantification confirmed a substantially higher myocyte viability after isolation from myocardial slices.Human cells, isolated with the described protocol, can be subjected to structural analysis. Alpha actinin staining, revealed a dense regular Z line pattern and a mean sarcomere length of 1.92 micrometers, in resting cardiomyocytes. LTCC and RyR staining, showed clear cluster is co-localized near the cell membrane and T tubules.The isolated cardiomyocytes were excitable and could be used for studies on cellular electrophysiology or excitation contraction coupling. Action potential shape and duration, was in the range reported by others, for ventricular cardiomyocytes, from failing human hearts. The calcium transients recorded by Khan vocal line scanning, showed a clear upstroke after stimulation.And, an acceptable signal to noise ratio. Cardiomyocyte shortening by contraction can be seen from the deflection of the lower cell border. Due to the high number of isolators cardiomyocytes obtained by this protocol, cell culture's possible.This may allow for gene transfer, pharmacological testing and tissue engineering of human cardiomyocytes. An important application, is the verification of findings, from animal models in human cells. Heart failure is a clinical syndrome with very limited therapeutic options.Improved cardiomyocyte isolation techniques will help to identify cellular targets for diagnosis and future therapies.

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