Name: isolation of human ventricular cardiomyocytes from vibratome-cut myocardial slices
Uploaded: 2020-05-10T13:30:00.000+00:00
Duration: 11 min 19 s
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, anesthetized by injecting 100 mg/kg of thiopental-sodium intraperitoneally, and euthanized by cervical dislocation followed by thoracotomy and excision of the heart. Human cardiac tissue samples were collected from the left-ventricular apical core during implantation of mechanical assist devices, from septal myectomy, from tetralogy of Fallot corrective surgery, or from the free left-ventricular wall of explanted hearts. The following protocol describes the isolation from human ventricular tissue. The isolation of rat cardiomyocytes was performed accordingly, but with different enzymes (see&#160;Table of Materials). A schematic workflow of the protocol is illustrated in&#160;Figure 1.
1. Preparation of buffers, solutions, and enzymes
<ol>
	<li>Prepare buffers and solutions as listed in Table 1.</li>
	<li>Warm up solutions 1, 2, 3 and the modified Tyrode&#8217;s solution to 37 &#176;C. Store the cutting solution at 4 &#176;C until use. 
	NOTE: For 1&#8211;2 myocardial slices a total of approximately 15 mL, 8 mL, and 5 mL of solutions 1, 2, and 3 are required for the isolation in one 35 mm tissue culture dish. Scale up accordingly for simultaneous myocyte isolations in multiple dishes. Cutting solution can be frozen and kept at -20 &#730;C for several months.</li>
	<li>Weigh 1 mg of proteinase XXIV (see Table of Materials) into a prechilled 15 mL centrifuge tube and store on ice until use. This is for processing one sample. Scale up the amount accordingly. Do not mix the proteinase and collagenase.</li>
	<li>Weigh 8 mg of collagenase CLSI (see Table of Materials) into a prechilled 15 mL centrifuge tube and store on ice until use. This is for processing one sample. Scale up the amount accordingly. Do not mix the proteinase and collagenase. 
	CAUTION: Wear a face mask or work under a fume hood to avoid inhalation of enzyme powder. 
	NOTE: Enzyme activity may vary in different lots. Therefore, the optimal concentration may differ and should be determined with each newly purchased enzyme16.</li>
</ol>
2. Storage and transport of myocardial tissue
<ol>
	<li>Store and transport human cardiac samples in cooled, 4 &#730;C cutting solution (Table 1).</li>
	<li>Use the same solution for further tissue processing and vibratome slicing. 
	NOTE: Biopsies and surgical heart samples should be transferred immediately to the cutting solution at 4 &#176;C and can then be stored or transported at 4 &#176;C for a maximum of 36 h before the application of this protocol.</li>
</ol>
3. Processing and slicing of the tissue
NOTE: The protocol for tissue slicing follows Fischer et al.15.
<ol>
	<li>Trimming of the tissue block 
	CAUTION: Human cardiac tissue is potentially infectious. Always use protective wear and follow safety regulations of your institution. Carefully handle used blades and discard in safety containers.
	<ol>
		<li>Place the specimen into a 100 mm tissue culture dish filled with 20 mL cold cutting solution and keep on a cooled, 4 &#176;C plate.</li>
		<li>Remove excess fibrotic tissue and epicardial fat with a scalpel. In case of a transmural specimen, remove trabeculae and tissue layers near the endocardium. 
		NOTE: Fibrotic tissue is stiff and appears white. Fat is typically soft and appears white to yellow. Trabeculae and endocardial tissue layers can be identified from their loose tissue composition and a nonaligned fiber orientation compared to myocardium of the epicardial layers</li>
		<li>For optimal vibratome processing, cut rectangular tissue blocks of approximately 8 mm x 8 mm x 8 mm with a scalpel from a larger tissue specimen. For smaller biopsies skip this step and move to agarose embedding.</li>
	</ol>
	</li>
	<li>Embedding cardiac tissue into low-melting-point agarose
	<ol>
		<li>Boil 400 mg of low-melting point agarose in 10 mL of cutting solution in a glass beaker. 
		CAUTION: Wear gloves and safety glasses to avoid burns. Handle hot glassware only with heat protection wear.</li>
		<li>Fill a 10 mL syringe with the hot, dissolved agarose gel. Seal the syringe and allow the agarose to equilibrate in a 37 &#176;C water bath for at least 15 min.</li>
		<li>Use forceps to place the trimmed cardiac specimen or biopsy into a clean 35 mm tissue culture dish with the epicardium facing down and remove excess fluid with a sterile swab.</li>
		<li>Pour the equilibrated agarose (step 3.2.2) over the tissue by emptying the syringe. Secure the tissue against movement with forceps while pouring the agarose. Make sure that the tissue is completely immersed in agarose.</li>
		<li>Immediately place the dish on ice and let the agarose solidify for 10 min.</li>
	</ol>
	</li>
	<li>Slicing the myocardium
	<ol>
		<li>Mount a new&#160;razor blade to the blade holder of the vibratome and calibrate the vibratome by adjusting the z-deflection of the blade if possible. 
		NOTE: This protocol uses a vibratome with an infrared-assisted calibration device to align the blade in a horizontal position with minimal z-deflection (measured deflection &#60;0.1 &#181;m).</li>
		<li>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 (agarose margins &#8805; 8 mm).</li>
		<li>Fix the agarose block to the specimen holder with a thin layer of cyanoacrylate glue and gentle pressure.</li>
		<li>Place the specimen holder with the tissue into the vibratome bath. Fill the bath with cutting solution and keep at 4&#8211;6 &#176;C throughout the processing with crushed ice, filled in the outer cooling tank of the vibratome.</li>
		<li>Generate 300 &#181;m thick slices with an advancing speed of &#8804;0.1 mm/s, an oscillating frequency of 80 Hz, a lateral amplitude of 1.5 mm, and a blade angle of 15&#176;. When handling the slices, hold the agarose instead of the tissue itself to avoid tissue damage. Store the slices in the cutting solution at 4 &#176;C for a maximum of 2 h, if necessary. 
		NOTE: Cardiomyocytes are oriented in parallel to the epicardium. Therefore, it is important to cut in parallel to the epicardium to avoid excessive myocyte damage. It is recommended to discard the first 1&#8211;3 slices, as only uniform slices of constant thickness should be used for the isolation. For small tissue biopsies, however, only discard the first slice.</li>
		<li>Check cardiomyocyte alignment under a standard light microscope with a magnification of 40-100x.</li>
	</ol>
	</li>
</ol>
4. Tissue digestion
<ol>
	<li>Place the heat plate on the lab shaker and warm it to 37 &#176;C. Start the lab shaker at 65 rpm.</li>
	<li>Dissolve the proteinase (prepared in step 1.3) in 2 mL of solution 1 (step 1.1 and 1.2) and incubate at 37 &#176;C until use. Do not mix the proteinase and collagenase.</li>
	<li>Dissolve the collagenase (prepared in step 1.4) in 2 mL of solution 1 (step 1.1 and 1.2) and incubate at 37 &#176;C until use. Do not mix the proteinase and collagenase. 
	CAUTION: Wear protective eyewear and gloves, because dissolved enzymes can cause skin and eye injuries.</li>
	<li>Add calcium chloride (CaCl2) to the collagenase containing solution (prepared in step 4.3) to a final concentration of 5 &#181;M.</li>
	<li>Use forceps to transfer a tissue slice from the vibratome bath to a clean 60 mm tissue culture dish filled with 5 mL of prechilled cutting solution (step 1.1 and 1.2) and keep on ice.</li>
	<li>Carefully remove the agarose from the myocardial tissue with blade or forceps. 
	NOTE: Avoid excess tension and shear stress on the myocardial slices, because they can damage the cardiomyocytes.</li>
	<li>To perform the initial wash, place a clean 35 mm tissue culture dish on the heat plate and fill it with 2 mL of prewarmed (37 &#176;C) solution 1. Transfer 1&#8211;2 myocardial slices to the prepared dish with forceps. Aspirate the solution with a 1 mL pipette and perform the wash steps 2x to remove remnants of cutting solution. 
	NOTE: Do not aspirate the cardiac slices. The solutions and the slices should remain at a constant temperature of 35 &#176;C on the agitated heat plate. Adjust the heat plate temperature if necessary.</li>
	<li>Remove solution 1 from the dish and add 2 mL of the proteinase solution (step 4.2). Incubate for 12 min on the heat plate at 65 rpm.</li>
	<li>Wash 2x with 2 mL of prewarmed solution 1 (37 &#176;C).</li>
	<li>Remove solution 1 from the dish and add 2 mL of collagenase solution (steps 4.3 and 4.4). Incubate at least 30 min on the heat plate at 65 rpm.</li>
	<li>Check for free individual myocytes at 30, 35, 40 min, etc., by placing the dish under a light microscope. Work quickly to avoid significant cooling of the solution. 
	NOTE: The required digestion time may vary depending on the tissue constitution and the degree of fibrosis. As soon as the tissue gets visibly soft and dissociates readily when gently pulled, the optimal digestion time has been reached. If enough tissue is available, several slices can be digested in parallel with varying digestion times.</li>
	<li>When the tissue is digested and individual myocytes are visible (step 4.11), wash 2x with 2 mL of prewarmed solution 2 (37 &#176;C) and fill again with 2 mL.</li>
</ol>
5. Tissue dissociation
<ol>
	<li>Dissociate the digested tissue slices with forceps by carefully pulling the fibers apart.</li>
	<li>Carefully pipette several times with a single-use Pasteur pipette (opening diameter &#62;2 mm). 
	NOTE: The use of forceps and pipetting can induce mechanical stress and cause cardiomyocyte damage. However, it is a crucial step for separation of the cells. Use fine forceps to minimize cell damage and carefully dissociate the slices to smaller pieces.</li>
	<li>Check for the liberated rod-shaped cardiomyocytes under the light microscope.</li>
</ol>
6. Reintroduction of physiological calcium concentration
<ol>
	<li>Slowly increase the calcium concentration from 5 &#181;M to 1.5 mM while agitating at 35 &#176;C on the heat plate. Use 10 mM and 100 mM CaCl2 stock solutions. Recommended steps: 20, 40, 80, 100, 150, 200, 400, 800, 1,200, 1,500 &#181;M. Allow the cells to adapt to the increased calcium levels in 5 min incubation intervals between each step.</li>
	<li>Remove undigested tissue chunks carefully with forceps or filter the cell suspension through a nylon mesh with 180 &#181;m pore size at the end of the calcium increase.</li>
</ol>
7. Removal of mechanical uncoupling agent
<ol>
	<li>Stop agitation and slowly remove one third of the solution (~700 &#181;L) from the top with a 1,000 &#181;L pipette. Avoid aspiration of the cardiomyocytes. 
	NOTE: If undigested tissue was removed, the cardiomyocytes will accumulate in the center of the dish, which facilitates aspiration of cell-free solution. If not, transfer the cell solution to a 15 mL centrifuge tube and allow the cells to sediment for 10 min at 35 &#176;C, then aspirate 700 &#181;L of the supernatant and discard, resuspend the cells, transfer them back to a 35 mm tissue culture dish and proceed with step 7.2.</li>
	<li>Add 700 &#181;L of solution 3 to the cells, resume agitation on the heat plate and incubate for 10 min.</li>
	<li>Repeat steps 7.1 and 7.2.</li>
	<li>Transfer the solution to a 15 mL centrifuge tube and allow the cardiomyocytes to sediment for a minimum of 10 min and a maximum of 30 min at room temperature or spin at 50 x g for 1 min. Remove the supernatant completely and resuspend in modified Tyrode&#8217;s solution or the desired experimentation buffer. 
	NOTE: Cardiomyocytes can be stored in modified Tyrode&#8217;s solution (Table 1) for several hours at 37 &#176;C and 5% CO2 before use.</li>
	<li>Verify the cell quality with a standard light microscope at 40x and 200x magnification. 
	NOTE: Around 30&#8211;50% of the cardiomyocytes should be rod-shaped, smooth without membrane blebs, and display clear cross-striations. Only 5&#8211;10% of the viable cells should show spontaneous contractions.</li>
</ol>

The authors have nothing to disclose.

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 this method, due to their small size or the absence of coronary artery branches, which makes new methods for the isolation of human cardiomyocytes desirable. The protocol described here is based on the atraumatic generation of thin cardiac tissue slices, which are then subjected to enzymatic digestion. It can be applied to myocardial specimens from animal hearts and human myocardium such as apical cores from left-ventricular assist-device implantation, endomyocardial biopsies from septal myectomy, or explanted hearts (see Supplemental Table 1), and reliably produces sufficient amounts of viable, calcium-tolerant cardiomyocytes that can be used for subsequent experiments, like cardiomyocyte cultivation17.
A major reason for higher yields of myocytes from vibratome-cut tissue slices than from minced tissue chunks might be a lower degree of myocyte damage during the slicing. When coronary perfusion is not possible, slicing or mincing the tissue specimen is necessary to increase the contact surface for digesting enzymes. Slice dimensions of approximately 8 mm x 8 mm x 0.3 mm allow for good access of enzymes due to a relatively large surface-to-volume ratio (~7 mm-1). Using scissors, a similar surface-to-volume ratio (~6 mm-1) can be achieved by mincing the specimens into small pieces of approximately 1 mm x 1 mm x 1 mm. Although myocyte damage in slices and minced chunks before enzymatic digestion was not quantified, slicing on a vibratome likely causes considerably less damage because it enables gentle cutting in the fiber direction. In fact, it was estimated that in vibratome-cut slices fewer than 5% of the myocytes are damaged19. The results show that human cardiomyocytes can be isolated very efficiently with the protocol provided, with a much higher proportion of viable cells compared to tissue chunks. This is in accordance with a protocol that used slices from atrial tissue20. Our method yields up to 200 calcium-tolerant ventricular myocytes per milligram of tissue and provides the possibility to store or transport cardiac tissue for up to 36 h prior to isolation. This renders the protocol applicable for laboratories without on-site cardiac surgery and thereby extends the possibilities for collaborations.
Critical steps in the protocol are the correct processing of myocardium to slices on a vibratome and their digestion as well as the final steps of calcium-reintroduction and washout of BDM. In contrast to other methods19,21, the cardiac tissue is embedded in agarose with a low melting temperature to avoid motion during the slicing procedure14,15. This was necessary to stabilize the tissue block and to generate uniform slices. When embedding the tissue, the agarose should still be liquid, but the temperature should not exceed 37&#8211;38 &#176;C to avoid cell damage by hyperthermia. Conversely, if the agarose is too cold (&#60;35 &#730;C), it does not bind well to the tissue block and may break during the slicing. To test for myocyte viability after the vibratome processing, a simple MTT assay that allows for quick evaluation of cell viability by light microscopy and also for quantification by photometrical analysis is suggested14,22. Cardiomyocyte damage can also occur during reintroduction of extracellular calcium after a period of incubation in calcium-free solution23,24. This calcium paradox phenomenon in isolated cells can be mitigated by a slow, stepwise increase of the calcium levels to allow the cardiomyocytes to adapt. This step should be carried out carefully during continuous agitation at 35 &#176;C and with intervals of 5 min. Slight hypothermia (21 &#176;C) increases the calcium tolerance of rat cardiomyocytes during reintroduction, which is in accordance with earlier reports25. However, human cardiomyocytes did not show major differences in calcium tolerance at 35 &#176;C or 21 &#176;C. The use of BDM during cutting, digestion, and reintroduction of calcium prevents myocyte contraction and cellular energy expenditure as well as hypercontraction and is therefore protective. However, for subsequent experiments assessing cardiac contractility or excitation-contraction coupling, BDM needs to be removed26. A gradual washout was applied to minimize spontaneous hypercontractions. BDM can be omitted, but this will markedly increase the amount of hypercontracted myocytes, as observed in previous experiments and studies6.
The described protocol uses a solution that contains high potassium and only traces of sodium for digestion. This solution gave the highest yields of rod-shaped, cross-striated cardiomyocytes. For enzymatic digestion, however, it required a higher concentration of collagenase (4 mg/mL) compared to digestion in normal Tyrode&#8217;s solution (1.5 mg/mL). High extracellular sodium may lead to intracellular sodium overload, exacerbating the negative effects of the calcium paradox, thus reducing cell yields27. Therefore, it is suggested to use solutions with high potassium and low sodium. A high extracellular magnesium concentration is frequently employed in cardiomyocyte isolation protocols3,28, and has been shown to reduce ischemia-reperfusion injury29,30 and improve cardiac function after extended periods of cold ischemia31. Therefore, the solution applied here also contains a high concentration of magnesium (10 mM). However, it was shown that excitability, contraction force, and conduction velocity may be reduced by high magnesium concentrations32. Although these effects were shown to be reversible, effects on isolated cardiomyocytes cannot be excluded. Thus, using magnesium at physiological concentrations (1&#8211;2 mM), might result in a lower overall cell yield but improve excitability.
The optimal digestion time is quite variable, as the amount of extracellular matrix or fibrosis can vary considerably in human failing myocardium. Should the tissue still be firmly connected at the time of dissociation, with only few viable myocytes released, increasing the digestion time in steps of 5 min and regularly checking for free myocytes and the texture of the slices is recommended. If the tissue remains undigested after prolonged periods (&#62;45 min), increasing the concentration of collagenase in steps of 1 mg/mL or testing a different enzyme batch is recommended16. The respective values presented in this work can serve as a primer for a robust isolation of cardiomyocytes, but the conditions for optimal yield and viability may be refined in each laboratory, taking into account the great variability of enzyme activities and tissue constitutions. An advantage of the method is that due to the small amounts of required tissue and the simple workflow in small batches, several tests can be carried out in parallel. Thus, an optimal protocol can be achieved quickly.
The generation of high-quality myocardial slices requires a high precision vibratome. The need for a high-precision tissue slicer or vibratome is a possible drawback of the method, because it is not easily available at every laboratory. The device used for this study is described in more detail elsewhere14,15. Different devices have been used successfully to generate myocardial slices by other groups33,34,35. Hence, if the settings of advancing speed, blade amplitude, and oscillation frequency can be met and a horizontal blade orientation with a z-deflection below 0.1 &#181;m is available, successful slicing and isolation should be feasible with other vibratomes. In addition, the tissue slicing is elaborate and significantly prolongs the duration of the protocol (approximately 1&#8211;2 h). The use of BDM as a mechanical uncoupling agent has protective effects by reducing cellular energy exposure, ischemic injury, and hypercontraction26,36,37. Although the negative inotropic effect of BDM was shown to be quickly reversible after washout38,39, it is not clear if BDM may have unknown consequences on cardiomyocyte function40. This study shows that cells can be isolated with high yields after tissue storage times of up to 36 hours and that human cardiomyocytes can be cultured for up to three days after isolation with this method17. We did not notice functional or structural differences between myocytes with short and long storage times. However, this was not assessed systematically. On the other hand, for whole rabbit hearts, it was shown that functional differences between fresh hearts and hearts exposed to cold ischemia in extracellular solution containing 30 mM&#160;BDM were negligible31, and the same may be true in this case.
The presented protocol provides a solid basis for all kinds of experiments with isolated ventricular cardiomyocytes and might be especially valuable for research on human myocardial specimens. With some adaptations, the isolation of other cardiac cells, such as fibroblasts or endothelial cells also seems possible41. Because it requires only small amounts of tissue that can be shipped in cold storage solution from other laboratories, application of the protocol may reduce the number of sacrificed laboratory animals. In fact, the protocol has been successfully applied to rabbit and porcine cardiac tissue. Furthermore, as experiments with long-term cultivated myocardial tissue slices increase21,33,42 and are constantly improved to provide optimal culture conditions15,43,44, the method can be readily applied after slice cultivation. Single cell isolation from cultivated myocardium may therefore help to characterize changes in cardiomyocytes after pharmacological or physical interventions in vitro.

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>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2,3-butanedionemonoxime</td><td>Carl Roth</td><td>3494.1</td><td>Purity&gt;99%</td></tr><tr><td>Bovine serum albumin</td><td>Carl Roth</td><td>163.2</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt; x 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>A537.1</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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>&lt;strong&gt;Dyes&lt;/strong&gt;</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>&lt;strong&gt;Enzymes&lt;/strong&gt;</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&lt;br/&gt; (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&lt;br/&gt; (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&lt;br/&gt; (activity &amp;ge; 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&lt;br/&gt; (activity: 7-14 U/mg)</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</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&amp;uuml;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 &amp;ge;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 &amp;micro;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.

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 ...

isolation-human-ventricular-cardiomyocytes-from-vibratome-cut

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Views.  Friedrich-Alexander University Erlangen-Nürnberg. 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 (

Video: Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Medicine

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 &#181;m are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode&#39;s solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 &#176;C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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.

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, ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.