Name: A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes (Video) | JoVE Journal
Uploaded: 2022-11-11T12:30:00.000+00:00
Duration: 6 min 25 s
Description: Learn a reliable non-Langendorff method to isolate mouse cardiomyocytes with minimal ischemic time using in vivo perfusion. This technique improves yield and requires less training than traditional approaches.

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5 minutes

This work was supported by National Institutes of Health Grants R01 HL114940 (Biesiadecki), R01 AG060542 (Ziolo), and T32 HL134616 (Sturgill and Salyer).

All procedures performed in this study were approved by the Institutional Animal Care and Use Committee at the Ohio State University in accordance with NIH guidelines.
1. Solution preparation
NOTE: Please see Table 2 for buffer concentrations.
<ol>
	<li>Pre-isolation (up to 2 weeks in advance of myocyte isolation)
	<ol>
		<li>Perfusion buffer base: Prepare 1 L of perfusion buffer base (NaCl, KCl, NaH2PO4, HEPES, Glucose, and BDM) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to 0.22 &#181;m sterile filtration. Store at 4 &#176;C until the day of isolation.</li>
		<li>Clearing buffer: Prepare 1 L of clearing buffer (NaCl, KCl, NaH2PO4, HEPES, Glucose, EDTA, and BDM) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to 0.22 &#181;m sterile filtration. Store at 4 &#176;C until the day of isolation.</li>
		<li>100 mM CaCl2 stock solution: Weigh out 1.11 g of CaCl2 and dissolve into 100 mL of ultrapure 18.2 M&#937;&#183;cm H2O. Store at room temperature until the day of isolation.</li>
		<li>Liberase aliquots: Dissolve 5 mg of digestive enzymes in 5 mL of sterile, RNase-free water. Prepare 0.8 mL aliquots and store at -20 &#176;C until the day of isolation.</li>
		<li>Laminate the dishes: Apply 100 &#181;L of 40 &#181;g/mL mouse laminin to sterile, glass-bottom 30 mL Petri dishes. Allow to sit for 30 min and remove excess laminin. Set the laminin on a Petri dish with UV incubation at room temperature for 30 min. Store at 4 &#176;C until the day of isolation (laminated Petri dishes can be used for up to 2 weeks).</li>
		<li>DMEM media: In a biosafety cabinet, add 2.5 mL of FBS, 0.5 mL of Penicillin-Streptomycin (50 U/mL-50 &#181;g), and 1 mL of 500 mM BDM to 46 mL of DMEM. Store at 4 &#176;C until the day of isolation (media can be used for up to 2 weeks).</li>
		<li>M199 media: Prepare 1 L of M199 media (NaHCO3, HEPES, L-glutathione, M199, BDM, and BSA) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to sterile filtration (0.22 &#181;m filter). Store at 4 &#176;C until the day of isolation.</li>
		<li>500 mM BDM stock: Add 0.5 g of BDM to 10 mL of ultrapure 18.2 M&#937;&#183;cm H2O. Store at 4 &#176;C until the day of isolation.</li>
		<li>Stabilizing platform creation: Mold playdough around the base of the needle screwed into the Luer stopcock. Mold to the Petri dish that will contain the heart and ensure that the needle is at proper height for ventricle injection (~5 mm above bottom of dish).</li>
	</ol>
	</li>
	<li>The day of isolation
	<ol>
		<li>Perfusion buffer: On the day of the isolation, add 9.5 mg of MgCl2 to 100 mL of perfusion buffer base. Allow to mix completely before removing 30 mL of completed perfusion buffer and place in a clean, wide-mouth, 100 mL glass bottle with a screw cap lid (digestion buffer). 
		&#8203;NOTE: These additions can be made on the day of isolation without additional pH adjustments as their addition negligibly alters the pH.
		<ol>
			<li>Remove 12 mL of perfusion buffer and set to the side (stop buffer). Pour the remaining 58 mL of perfusion buffer into another clean, wide-mouth, 100 mL glass bottle with a screw cap lid. Store the remaining perfusion buffer at 37 &#176;C throughout isolation.</li>
		</ol>
		</li>
		<li>Digestion buffer: Add 7.5 &#181;L of 100 mM CaCl2 to 30 mL of perfusion buffer for a final concentration of 25 &#181;M. Immediately prior to the isolation, add 770 &#181;L of Liberase to the digestion buffer for a final concentration of 26 &#181;g/mL.</li>
		<li>Stop buffer: Dissolve 240 mg of BSA in 12 mL of perfusion buffer. Store at 37 &#176;C throughout isolation.</li>
		<li>Clearing buffer: Prepare a 3 mL syringe of clearing buffer and clear the needle of bubbles. Store on ice until isolation.</li>
	</ol>
	</li>
</ol>
2. Manifold preparation
<ol>
	<li>Clear the temperature-controlled manifold with freshly prepared perfusion buffer, being careful to ensure no bubbles remain. Using a Luer lock connector, screw in a 27 G needle, and clear of bubbles. A cleared manifold is essential for a successful isolation.</li>
</ol>
3. Animal preparation
<ol>
	<li>Inject the mouse intraperitoneally with 100 mg/kg ketamine and 20 mg/kg xylazine by body weight immediately prior to the isolation. This procedure used a male, 4-month-old C57Bl/6 mouse.</li>
	<li>If needed, place the mouse on a heated surgical pad to encourage sedation. Tent the arms of the mouse, and tape the limbs and base of the tail to a blue lab diaper. Ensure that the animal is fully anesthetized&#160;by the withdrawal of&#160;toe-pinch reflex. Apply a sterile drape around the surgical area.</li>
</ol>
4. Cardiomyocyte isolation procedure
<ol>
	<li>Expose the sternum of the mouse and, lateral to the midline, cut proximally through the ribs and to the axilla. Gently cut fully through the diaphragm, ensuring shallow cuts to avoid the heart. Clamp the sternum with a hemostat and fold the ribs backwards to expose the thoracic cavity.</li>
	<li>Gently remove the pericardium from the heart and cut fully through the inferior vena cava, immediately distal to the heart. Use a 3 mL syringe with a 27 G needle to quickly inject 3 mL of ice-cold clearing buffer into the right ventricle of the heart over 1 min.</li>
	<li>Gently hold the heart using tweezers and pull away from the body, exposing as much of the aorta as possible. Using a hemostat, clamp the ascending aorta, being careful not to clamp the atria, and excise the heart from the chest.</li>
	<li>Quickly transfer the clamped heart to the lid of a polypropylene Petri dish with approximately 10 mL of warm perfusion buffer. Place the clamped heart into the supporting platform and use a stabilized 27 G needle attached to the manifold to inject 10 mL of temperature-controlled 37 &#176;C perfusion buffer into the left ventricle over 5 min.</li>
	<li>Transfer the clamped heart and supporting platform to a Petri dish with approximately 5 mL of digestion buffer. Exchange input syringes for a 50 mL syringe with 25 mL of digestion buffer.</li>
	<li>Clear the manifold of any bubbles and remaining perfusion buffer before injection. Carefully replace the needle into the same needle position in the left ventricular apex.</li>
	<li>Use a perfusion pump to inject temperature-controlled 37 &#176;C digestion buffer into the left ventricle for 15 min using a 50 mL syringe. Control the temperature of the solution with a water jacket previously calibrated to eject 37 &#176;C solution at the end of the needle.</li>
	<li>Remove the ventricles from the atria and transfer to a 10 mL beaker. Add 3 mL of digestion buffer to the beaker and, using sharp scissors, cut the ventricles into large chunks. Cover the beaker with aluminum foil and place in a shaking water bath preheated to 37 &#176;C for 5 min.</li>
	<li>Discard the supernatant, being careful to avoid removing any chunks of tissue. Resuspend the tissue chunks in 3 mL of digestion buffer and triturate for approximately 4 min or until a homogenous mixture is achieved.</li>
	<li>Filter the cells through a 70 &#181;m nylon cell strainer into a 50 mL polypropylene tube.</li>
	<li>Return the filtrate to a 14 mL round-bottom polypropylene tube and centrifuge for 1 min at 100 rpm. Discard the supernatant and resuspend the pellet in 3 mL of stop buffer.</li>
	<li>Add 54 &#181;L of 100 mM CaCl2 stock to the resuspended cells for a final CaCl2 concentration of 1.8 mM. This step can also be performed stepwise to increase cell yield.</li>
	<li>Allow live cells to settle by gravity for 10 min before removing the supernatant containing dead cells. Resuspend the pellet in storage solution (perfusion solution with 200 &#181;M CaCl2). These cells can now be used for functional experiments (i.e., calcium imaging/contraction, patch clamp, etc.), culture, etc. 
	&#8203;NOTE: The cells may also be resuspended in lab- or experiment-dependent solutions.</li>
</ol>
5. Cell culture
<ol>
	<li>Count the cells using a hemocytometer or by a field view count using a gridded cover slip. As myocytes are very large, counting using a hemocytometer is not always accurate. This is used to get an idea of approximately how many cells were obtained from the isolation.</li>
	<li>Dilute the cells to ~25,000 cells/mL with DMEM media. Add 1 mL of cell solution to each well.</li>
	<li>Incubate at 37 &#176;C, 95% O2, 5% CO2 for 2 h.</li>
	<li>Gently aspirate DMEM media from each well and add 2 mL of M199 media.</li>
	<li>Incubate at 37 &#176;C, 95% O2, 5% CO2 for up to 24 h.</li>
</ol>

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</ol>

No conflicts of interest to disclose.

The principal advantage of our Langendorff-free cardiomyocyte isolation technique is that it limits hypoxia and ischemic time by not requiring cannulation to a Langendorff apparatus. Alternatively to classical Langendorff techniques that take several minutes to remove, clean, and hang the heart, often resulting in ischemic damage to the myocyte, our method includes an in vivo clearing of blood via an ice-cold clearing solution. The ice-cold clearing buffer contains ethylenediaminetetraacetic acid (EDTA)...

For decades, an essential idea in cardiac biology literature is the molecular mechanism of action. The mechanism of action must be established in order to publish reliable studies. A well-established strategy to determine molecular mechanism is isolated cardiomyocyte studies, which require high-quality cardiomyocytes for attaining trustable data. Cellular and molecular experiments performed on cardiomyocytes to determine mechanism of action are the gold standard for investigating contraction1, electrophysiology2, calcium (Ca2+) cycling3, myofilament Ca2+ sensitivity4, cytoskeleton5, metabolism6, effects of hormones7, signaling molecules8, drug studies9, etc. The mouse has become the species of choice for most cardiac biology experiments due to the ease of genetic manipulation, its small size, its relatively short lifespan, low cost, etc10. However, the reliable isolation of high-quality mouse cardiomyocytes is not trivial with current techniques.
Labs have been isolating cardiomyocytes for almost 70 years11. Virtually all techniques to isolate cardiomyocytes rely on digestion of the heart via various enzymes (collagenase, protease, trypsin, etc.). In the early periods (1950s-1960s), the chunk method was employed, which involved removing the heart, cutting into much smaller pieces and incubating in solution with collagenase/protease/trypsin12. In the 1970s labs implemented the ameliorated &#8220;Langendorff&#8221; method13, which isolated cardiomyocytes using a coronary artery perfusion-based isolation technique (retrograde perfusion with enzyme via the Langendorff apparatus); this technique remains the dominant method of myocyte isolation in the field today, ~50 years later14,15,16. Recent work has shifted to cannulating the heart in vivo to limit hypoxia time and ischemic damage resulting in superior cardiomyocyte isolations (better yields and higher quality)17. Recently, this has evolved into performing in vivo, Langendorff-free heart perfusions18,19,20,21,22. We have evolved the Langendorff-free cardiomyocyte isolation technique based on the Ackers-Johnson et al.18 technique and adapted various components from the many previous isolation techniques. These key adaptations include the injection of an ice-cold clearing buffer and the incorporation of a supporting platform to stabilize the needle, allowing for decreased manipulation of the heart. Also detailed in this technique is&#160; temperature control of injected buffers (37 &#176;C), which decreased the time between in vivo injection and digestion due to less EDTA perfusion as previously published18. By decreasing manipulation of the heart and therefore minimizing puncture site size, thorough and constant perfusion of the coronary arteries is obtained. We also refined the technique with a secondary chunk method digestion,&#160;the amount of EDTA in the injected clearing buffer, and changed the pH. Our described technique is more reliable, more efficient, and does not require the extensive training/practice compared to the using the Langendorff apparatus (Table 1).

<table><tbody><tr><td>10 cc Bd Luer-Lok Syringe</td><td>Fisher Sci</td><td>14-827-52</td><td></td></tr><tr><td>10 mL Pyrex Low-Form Beaker</td><td>Cole-Palmer</td><td>UX-34502-01</td><td></td></tr><tr><td>100 mL polypropylene cap glass media storage bottle</td><td>DWK Life Sciences</td><td>UX-34523-00</td><td></td></tr><tr><td>14 mL Round-Bottom Polypropylene Test Tubes With Cap</td><td>Fisher Sci</td><td>14-959-11B</td><td></td></tr><tr><td>2,3-Butanedione Monoxime</td><td>Sigma</td><td>B0753</td><td>&gt;98%</td></tr><tr><td>3 cc BD Luer-Lok Syringe</td><td>Fisher Sci</td><td>14-823-435</td><td></td></tr><tr><td>35 mm glass bottom dishes</td><td>MatTek Corporation</td><td>P35G-1.0-20-C</td><td></td></tr><tr><td>50 mL BD Syringe without Needle</td><td>Fisher Sci</td><td>13-689-8</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Cole-Palmer</td><td>EW-22999-84</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt; 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>AIMS Space Gel Heating Pad</td><td>Fisher Sci</td><td>14-370-223</td><td></td></tr><tr><td>BD PrecisionGlide 27 G X 1/2&quot; Hypodermic Needles</td><td>Becton Dickinson</td><td>305109</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3803</td><td>Heat shock fraction, lyophilized powder, essentially fatty acid free, &gt;98%</td></tr><tr><td>Calcium Chloride dihydrate</td><td>Sigma</td><td>C7902</td><td>&gt;99%</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td>Suitable for cell culture, &gt;99.5%</td></tr><tr><td>DMEM</td><td>Fisher Sci</td><td>11965092</td><td></td></tr><tr><td>EDTA</td><td>Fisher Sci</td><td>AAA1071336</td><td></td></tr><tr><td>Falcon 100 mm TC-treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>FBS</td><td>R&amp;amp;D Systems (Bio-techne)</td><td>S11195</td><td></td></tr><tr><td>Fisherbrand Isotemp Heated Immersion Circulators</td><td>Fisher Sci</td><td>13-874-432</td><td></td></tr><tr><td>Hartman Mosquito Hemostatic Forceps</td><td>World Precision Instruments</td><td>15921</td><td></td></tr><tr><td>Hausser Scientific Hy-Lite Counting Chamber Set</td><td>Fisher Sci</td><td>02-671-11</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>&gt;99.5%</td></tr><tr><td>Labeling Tape</td><td>Fisher Sci</td><td>15-901-10R</td><td></td></tr><tr><td>Legato 100 Syringe Pump</td><td>kdScientific</td><td>788100</td><td></td></tr><tr><td>L-glutathione</td><td>Fisher Sci</td><td>ICN19467980</td><td></td></tr><tr><td>Liberase TH Research Grade</td><td>Sigma</td><td>5401135001</td><td>High thermolysin concentration</td></tr><tr><td>M199</td><td>Fisher Sci</td><td>MT10060CV</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Invitrogen</td><td>AM9530G</td><td></td></tr><tr><td>Mouse Laminin</td><td>Corning</td><td>354232</td><td></td></tr><tr><td>Pen/Strep</td><td>Fisher Sci</td><td></td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P5405</td><td>&gt;99%</td></tr><tr><td>Precision Digital Reciprocating Water Bath</td><td>ThermoFisher Scientific</td><td>TSCIR19</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td>Suitable for cell culture</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S5886</td><td>&gt;99%</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S5011</td><td>&gt;99%</td></tr><tr><td>Sterile Cell Strainer 70 &amp;micro;m</td><td>Fisher Sci</td><td>22-363-548</td><td></td></tr><tr><td>Student Fine Scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>VWR Absorbent Underpads</td><td>Fisher Sci</td><td>NC9481815</td><td></td></tr></tbody></table>

a simple and effective method to consistently isolate mouse cardiomyocytes

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

There are a few elements to examine when determining the success of an isolation. First, the cardiomyocytes must be rod-shaped with no membrane blebs, such as the cells isolated in Figure 1. A typical isolation will yield ~80% of the myocytes being rod-shaped. If the isolation yields anything less than 50% rod-shaped cells, then it is considered an unsuccessful isolation and cardiomyocytes are not used. Lastly, the cardiomyocytes should be quiescent. Spontaneously contraction myocytes demons...

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

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Research

JoVE Journal

Medicine

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system.  The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression.  Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation and Physiological Analysis of Mouse Cardiomyocytes

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

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.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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.

Isolation of Atrial Myocytes from Adult Mice

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

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.

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

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.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

Learn a reliable non-Langendorff method to isolate mouse cardiomyocytes with minimal ischemic time using in vivo perfusion. This technique improves yield and requires less training than traditional approaches.

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

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