Name: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes (Video) | JoVE Journal
Uploaded: 2016-08-28T13:00:00.000+00:00
Duration: 8 min 42 s
Description: Learn to isolate and culture adult mouse cardiomyocytes with high yield and viability for cardiovascular research. This method enables adenoviral transduction to study cell cycle regulation in cardiac cells.

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5 minutes

This project was funded by the UCSF Program for Breakthrough Biomedical Research (funded in part by the Sandler Foundation), the NIH Pathway to Independence Award (R00HL114738), and the Edward Mallinckrodt Jr. Foundation. JJ was supported by a postdoctoral fellowship from the NIH (T32HL007731). The authors are solely responsible for the contents of this work, which does not necessarily represent the official views of the NIH.

All procedures outlined here have been approved by the Institutional Animal Use and Care Committee at the University of California, San Francisco.
NOTE: Briefly, after extracting the heart from the mouse thorax, coronary retrograde perfusion is used to efficiently digest the extracellular matrix with collagenase and protease XIV. The ventricles are then isolated, mechanically dissociated and filtered into a single cell suspension. Gravity sedimentation is performed to isolate cardiomyocytes, which are separated from stromal cells like fibroblasts and endothelial cells by their high density. The purified cardiomyocytes can be cultured for weeks on laminin-coated polystyrene and transduced with adenovirus gene vectors.
1. Cardiomyocyte Isolation
<ol>
	<li>Prepare Buffers, Surgical Station and Perfusion Apparatus
	<ol>
		<li>Prepare buffers and media according to Table 1.</li>
		<li>Turn on circulating water bath, set input temperature such that output of heated water jacket reaches a temperature of 37 &#730;C.</li>
		<li>Clean perfusion system by flushing with 70% ethanol. Then remove all traces of ethanol from system by flushing with several volumes of sterile water.</li>
		<li>Load the perfusion system with perfusion buffer (Table 1). First drain water from system, then flush with 2 volumes of perfusion buffer. Then fill the dead space with perfusion buffer such that the inner column of the heat jacket is filled with perfusion buffer, but the debubbling chamber is empty. 
		TIP: Ensure no air bubbles are present in the line below the debubbling chamber. To remove recalcitrant bubbles, engage all stopcocks and allow pressure to build up inside the line for a few seconds. Then release the lower stopcock, allowing the high pressure perfusion buffer to jet from the outlet; the rapid flow and turbulence will help dislodge bubbles that are caught near the perfusion outlet.</li>
		<li>Prepare a syringe with needle to be used for aortic cannulation. Attach an 18 G blunt needle to a 1 ml syringe filled with perfusion buffer. Remove air bubbles and fix the syringe assembly to one arm of the dissecting miscroscope illuminator with lab tape. 
		TIP: To create a blunt ended needle from a beveled needle, transect the needle shaft with wire cutters. Then hone the tip with a sanding stone until the tip is flat. 
		NOTE: To reduce slippage of the aorta from the needle during cannulation, roughen the shaft of the cannulation needle using a razor blade. Secure the needle by the hub and upper shaft, then saw the razor back and forth perpendicular to the long axis of the needle shaft while rotating until several grooves have been created within 1 cm of the tip of the needle shaft. Switch to a new razor when the blade becomes dull.</li>
		<li>Place a clean petri dish below the dissecting microscope to contain the heart during cannulation. Position the illuminator arm such that the tip of the cannula is near the edge of the viewing field, but not obstructing the dissection. Ensure the tip of the cannula is below the rim of the petri dish. 
		NOTE: To help immobilize the heart during dissection, place a small (~ 5 - 7 cm2) square of 215 micron mesh in the petri dish. When the heart is placed on this mesh, it will create friction to reduce sliding and rotating of the heart during mechanical manipulation.</li>
		<li>Tie 2 sutures with loose double overhand knots around the hub of the cannula needle to be used for securement of the aorta.</li>
	</ol>
	</li>
	<li>Heart Extraction and Cannulation
	<ol>
		<li>Administer heparin (5 IU/g body weight) via intraperitoneal injection. Wait 20 min to allow the heparin to circulate to the heart.</li>
		<li>Anesthetize the mouse by intraperitoneal injection of 20% ethyl carbamate (2 mg ethyl carbamate/g body weight). Observe closely until the mouse is under deep anesthesia (approximately 3 - 5 min). 
		NOTE: Deep anesthesia is confirmed by a lack of both toe pinch and corneal reflexes. If the mouse has not reached deep anesthesia by 8 - 10 min, then administer an additional dose of ethyl carbamate. Although individual responses to anesthesia will vary between mice, failure to enter deep anesthesia upon first dosing may indicate degraded ethyl carbamate, which may preclude deep anesthesia even after repeated dosing. To prevent degradation, prepare a fresh solution of ethyl carbamate before each use.</li>
		<li>Immobilize the mouse to the surgery platform by securing each limb with surgical tape. Disinfect&#160;the incision area with a liberal amount of 70% ethanol. Pat dry with a lab-wipe.</li>
		<li>Lift the skin with tissue forceps just below the sternum. Make a lateral incision with small scissors, cutting through the skin and abdomen.</li>
		<li>Use hemostats to gently lift the ribcage via the sternum. Using small dissection scissors, extend the incision in a V-shape toward the axillae. Cut through the first rib and puncture the diaphragm on each side.</li>
		<li>Continue to lift the ribcage with hemostats and use small iris scissors to completely transect the diaphragm. Take care not to puncture the heart.</li>
		<li>Retract the ribcage rostrally using attached hemostats and quickly but carefully extend the V-shape incision by cutting through the ribcage toward the axillae on both sides. Retract the middle section of the ribcage fully and lay down the attached hemostats to hold in place.</li>
		<li>Remove the pericardium using fine forceps.</li>
		<li>While gently lifting the heart using curved forceps, transect the veins and arteries attached to the heart. Immediately place the heart in ice-cold perfusion buffer to slow muscle contraction.</li>
		<li>Place the heart in a petri dish under a dissection microscope (place on a small piece of 215 &#956;m mesh to help immobilize during dissection). Trim any excess non-cardiac tissue using fine iris scissors. Use caution when cutting near the aorta.</li>
		<li>Transect the aorta near the brachioencephalic artery, and take care not to let bubbles or tissue debris enter the opening.</li>
		<li>Fill the petri dish containing the heart with ice cold perfusion buffer such that the liquid level rises above the opening of the cannula. Sheath the aorta onto the cannula such that the tip is just distal to the aortic valve. 
		TIP: To reduce the risk of air embolism, slightly depress the syringe on the cannula prior to cannulation in order to remove potential air bubbles which may have developed at the end of the cannula. Additionally, keep the tip of the cannula and the aorta under the surface of the solution to avoid introducing bubbles while sheathing the aorta.</li>
		<li>Slide a pre-tied 7-0 silk suture down the shaft of the cannulation needle and position just above the tip of the needle. Tighten the knot with fine forceps. Repeat this step with a second suture placed just above the first. 
		NOTE: Ensure the tip of the needle is still above the aortic valve before and after tightening.</li>
		<li>Slowly depress the syringe to eject 0.5 ml of perfusion buffer through the coronary vasculature. 
		NOTE: If the cannulation was successful, blood will be visualized leaving the coronary vasculature and pooling out of the dorsal veins and arteries. 
		IMPORTANT: The time from opening of the thoracic cavity (in particular, puncturing the diaphragm) to initial perfusion should be minimized to reduce hypoxic exposure and maximize the survival and health of the isolated cardiomyocytes. Ideally, this time should be less than 5 min.</li>
	</ol>
	</li>
	<li>Perfusion 
	NOTE: The retrograde perfusion apparatus can be conveniently operated in a clean environment on a standard laboratory bench or placed in a laminar flow hood to maximize sterility. However, after perfusion is complete, work should be performed under laminar flow to minimize contamination.
	<ol>
		<li>Gently but firmly remove the cannula hub from the syringe (using sterile gloves) by slowly twisting back and forth. While pulling the cannulation needle off of the syringe, slowly eject perfusion buffer into the needle hub in order to prevent air bubbles during perfusion.</li>
		<li>Remove the recirculating catch tube from below the perfusion outlet port and replace with a waste catch beaker. Attach the needle hub to the luer adaptor that is fixed to the perfusion outlet port (Figures 1, 2) on the water jacket, while the pump is running at the lowest setting (15 RPM, ~ 6 ml /min). 
		TIP: When connecting the needle hub to the luer adaptor, allow the dripping perfusion buffer to connect to the liquid in the needle hub in order to avoid introducing bubbles.</li>
		<li>Perfuse the heart without recirculation until 8 ml of perfusion buffer have been aspirated by the inlet hose. 
		NOTE: The volume of perfusion buffer aspirated by the inlet hose at this step assumes a dead volume of 7 ml for the perfusion system, giving a total of 15 ml of perfusion buffer pumped through the heart.</li>
		<li>Remove the inlet tube from the perfusion buffer. Allow air to be aspirated by the inlet for less than the dead volume of the system. 
		NOTE: The volume of air aspirated in this step must be adjusted to prevent air from entering the coronary vasculature (see step 1.3.7).</li>
		<li>Place the inlet tube into the digestion buffer (Table 1), just above the bottom of the tube. Feed slowly and allow the inlet tube to aspirate enough volume to avoid overfilling the tube.</li>
		<li>Visualize and follow the air bubble between the perfusion buffer and the digestion buffer as it travels through the heat jacket. Observe the level of perfusion buffer in the center of the heat jacket begin to drop once the air bubble reaches the de-bubbling chamber. Once the digestion buffer reaches the de-bubbling chamber, the level of liquid in the heat jacket will remain steady. 
		IMPORTANT: If the level of perfusion buffer drops too low, close the outlet stopcock and open the vent stopcock. Allow the level to rise to a comfortable level (3 - 5 inches above the outlet port). Then, stop the perfusion pump and close the vent stopcock. Open the outlet port stopcock and restart the perfusion pump.</li>
		<li>Watch the solution emerging from the heart. As the last of the perfusion buffer leaves the heart and the digestion buffer begins circulating through the heart, color change from clear to light brown will be observed due to the presence of collagenase.</li>
		<li>Perfuse the heart with digestion buffer for approximately 10 - 15 min. The heart will begin slouching as the collagen is degraded and it loses mechanical support.</li>
	</ol>
	</li>
	<li>Mechanical Dissociation and Purification
	<ol>
		<li>Remove the heart from the cannula with forceps and place in a dry petri dish. Quickly trim extra-ventricular tissue using fine iris scissors. Transfer the ventricles to a fresh petri dish containing perfusion buffer to wash. Move the petri dish to a sterile hood and transfer the ventricles to a fresh, dry petri dish.</li>
		<li>Collect 7.5 ml of digestion buffer from the heat jacket and add 2.8 &#956;l of 1 M CaCl2, mix by inversion. Add 2 - 3 ml of digestion buffer with CaCl2 to the ventricles in the petri dish. 
		NOTE: The concentration of Ca2+ is raised from 300 to 700 &#956;M</li>
		<li>Quickly triturate the ventricular tissue with fine forceps, so that most pieces are smaller than 1 mm3. Add the remaining Ca2+ supplemented digestion buffer from step 1.3.2 to the dissociated ventricles in the petri dish and mix by gentle agitation.</li>
		<li>Place the petri dish in a 37 &#176;C incubator with 5% CO2. Agitate the petri dish every 2 min until sufficient cardiomyocytes (see NOTE below) have been released from the tissue. 
		NOTE: The length of incubation may need to be adjusted to fine tune the degree of digestion according to the needs of the experiment. Generally, longer incubation times increase yield and purity, but may reduce the rate of cell attachment and survival. 5 - 10 min is generally sufficient to achieve a high yield (~ 5 - 10 x 105 cells per heart).</li>
		<li>Transfer the petri dish back to a laminar flow hood. Using a transfer pipette cut at a 45&#176; angle, gently pipette the tissue to further dissociate.</li>
		<li>Use sterile gloves to fold the 215 &#956;m mesh into a cone and hold over a 50 ml conical. Pipette the dissociated tissue suspension onto the mesh and allow the filtrate to drain into the 50 ml conical. Use 7.5 ml of pre-warmed stop buffer (Table 1) to wash the cardiomyocytes from the petri dish through the mesh, combining with the first filtrate. 
		NOTE: Ca2+ is raised to final 1.1 mM and the final 2.5% FBS in the stop buffer neutralizes protease activity.</li>
		<li>Transfer the cell suspension to a 15 ml conical and allow to settle by gravity for 15 min.</li>
		<li>Carefully remove the supernatant with a transfer pipette such that only 50 - 100 &#956;l of solution remains above the cells. Add 10 ml of pre-warmed, equilibrated culture media (Table 1) and mix by gentle inversion. Allow the cardiomyocytes to settle by gravity for 15 min. 
		TIP: To increase purity, repeat step 1.4.8 as desired.</li>
		<li>Carefully remove the supernatant with a transfer pipette such that only 50 - 100 &#956;l of solution remains above the cells.</li>
	</ol>
	</li>
</ol>
2. Cardiomyocyte Culture
<ol>
	<li>Pre-coat tissue culture-treated petri dishes with laminin (10 &#956;g/ml) and store at 37&#176; C for 2 hr prior to plating cells. Just prior to plating, aspirate the laminin solution from the tissue culture plate. Wash once with MEM or PBS, then aspirate residual liquid to remove non-adsorbed laminin.</li>
	<li>Add enough pre-warmed, equilibrated culture media to achieve desired cell density, mix by gentle inversion. Plate desired concentration of cells into the culture vessel (typically 5 - 20 x 104 cells/cm2). To assess the yield of cardiomyocyte isolation, count the cells via hemacytometer or test-plate by checking the density under a microscope. An adult mouse heart typically provides 0.5 - 1.0 x 106 cells at this stage of the protocol. 
	TIP: If using 96 well plates, use a 1,000 &#956;l micropipette with a tip cut at a 45&#176; angle to minimize cell damage due to shear force. Place the pipette tip such that it is touching the bottom of the well during ejection of cell suspension in order to minimize introduction of bubbles.</li>
	<li>Move the cell culture dish promptly to a 37 &#176;C incubator with 5% CO2 and 95% humidity. Change media as needed to prevent acidification: every 1 - 5 days depending on cell density. Minimize handling of the culture dish during the first 3 - 6 days to facilitate the attachment to the cell culture surface. 
	NOTE: To minimize cell disturbance during media changes when the cells have not fully attached, perform a half-media change. Slowly aspirate the media while keeping the pipette tip just below the surface of the media. Then slowly add additional media by keeping the pipette tip just above the surface of the media against the wall of the culture dish.</li>
	<li>To assess the viability of cardiomyocytes, check the morphology under brightfield microscopy. Freshly isolated cardiomyocytes maintain a roughly rod-shaped morphology with sharp, distinct edges under brightfield illumination (Figure 2). 
	NOTE: Confirm viability by staining the cells with trypan blue.</li>
	<li>To assess the purity of the cardiomyocyte isolation, check the cell morphology to identify cardiomyocytes. Stain the nuclei with Hoescht 33342 to identify total cells. 
	NOTE: Cardiomyocytes can be identified from non-cardiomyocytes by their characteristic morphology (rod-shaped) and size (approximately 100 - 200 &#956;m in length). Alternatively, freshly isolated cardiomyocytes will stain positive for cardiomyocyte-specific markers, such as alpha-actinin26 (Figure 3C, D) or cardiac troponin T.27</li>
</ol>
3. Gene Transduction with Adenovirus
<ol>
	<li>Prepare adenovirus cell lysate using established methods.21 Transduce the cardiomyocytes using cell lysate from adenovirus producer cells 10. On day 0 or day 1 after isolation, carefully add the cell lysate to the media containing the cardiomyocytes. 
	NOTE: A high titer adenovirus preparation will achieve strong expression in approximately 48 - 72 hr. Adjust the multiplicity of infection to suit the experimental design.</li>
</ol>

The authors have nothing to disclose.

The overall health of the isolated cardiomyocytes depends on several important aspects of this protocol. First, the time from heart extraction to perfusion is critical and should be performed in 5 min&#160;or less. Removal of calcium helps to dissociate cell-cell interactions, but can negatively impact cell health long-term.29-32 Thus, we find it sufficient to remove calcium during the initial few minutes of perfusion by EGTA (ethylene glycol tetraacetic acid) chelation,22 but quickly restore calciu...

Cultured cardiomyocytes are frequently used to monitor cell behavior in a well-controlled environment in vitro. For example, morphological, electrical, biochemical, or mechanical cell properties can be studied on engineered substrates,1,2 in defined media, and in response to small molecule drugs, peptides, gene regulation,3 or electrical stimulation.4 The cellular content can also be controlled using defined co-cultures.5 These in vitro experiments are useful in large drug or genetic screens and complement in vivo methods for various types of investigations involving cardiomyocyte biology.
Long-term culture enables experimental avenues that require extended periods of time to achieve phenotypic change. A timely example is that of adult mammalian cardiomyocyte proliferation, where dedifferentiation, cell cycle re-entry, and cell division is typically studied over several days to weeks.6,7 Here, the extended culture time facilitates genetic manipulation,7,8 functional dedifferentiation (e.g., sarcomeric disassembly)9 and potentially transcriptional dedifferentiation.6 Subsequent cell cycle re-entry and cell division requires even longer culture periods to observe, especially if multiple rounds of division are the experimental goal. The importance of the cardiomyocyte cell-cycle is central to several recent key scientific works in heart regeneration, where the dedifferentiation and proliferation of pre-existing cardiomyocytes has been shown responsible for heart regeneration in zebrafish and neonatal mice.10-12 Thus, the possibility to stimulate dedifferentiation and cell cycle re-entry in mammalian adult cardiomyocytes remains a key question in human heart regeneration13-15
In vitro experiments studying the cell cycle of mammalian cardiomyocytes have predominantly used rat sources, due to their relative ease of long-term culture compared to mouse models.16 However, murine systems offer a rich resource of well-characterized transgenic tools and disease models that are useful in both in vitro and in vivo protocols. For example, Cre-based lineage tracing has enabled the identification of pre-existing cardiomyocytes as a source of regenerating myocardium in the neonatal mouse heart in vivo.12 In vitro studies of lineage-traced neonatal mouse cardiomyocytes have enabled the examination of interactions with stromal cells through co-culture with fibroblasts.5 However, due to its challenges,17 few reports exist of the isolation and long-term culture of adult mouse cardiomyocytes.18,19
The isolation of viable adult mouse cardiomyocytes for short-term culture alone is known to be a challenging task. This protocol provides step-by-step instructions on how to achieve viable cardiomyocytes from adult mice that can be used for both short-term as well as long-term investigations. Cardiomyocytes isolated using this protocol can be efficiently transduced with adenoviral vectors20,21 and cultured for weeks. These methods provide a powerful system to study cardiomyocyte biology in vitro.
The methods described herein are based on several elements from previous works using variations of the Langendorff retrograde perfusion.18,22 Although several protocols have been published on the isolation of adult mouse cardiomyocytes for short-term culture and study,23-25 the advantage of this protocol is the ability to culture the isolated cardiomyocytes long-term. This will be useful in the study of cellular processes involving ectopic gene expression and requiring extended periods of time, such as in cellular reprogramming strategies.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Heated water jacket</td><td>Radnoti</td><td>158831</td><td></td></tr><tr><td>Circulating heated water bath, Isotemp</td><td>Fisher Scientific</td><td>3013</td><td></td></tr><tr><td>Laboratory pump</td><td>Watson-Marlow</td><td>323</td><td></td></tr><tr><td>Hemostats</td><td>Exelta</td><td>63042-090</td><td></td></tr><tr><td>Tissue forceps</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Dumont #7 curved forceps</td><td>FST</td><td>91197-00</td><td></td></tr><tr><td>Dumont #5 fine forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Small dissection scissors</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Extra fine bonn scissors</td><td>FST</td><td>14084-08</td><td></td></tr><tr><td>Fine spring scissors</td><td>FST</td><td>91500-09</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25837</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25414</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>86329</td><td></td></tr><tr><td>Butane dione monoxime (BDM)</td><td>Sigma</td><td>B0753</td><td></td></tr><tr><td>HEPES</td><td>Fisher&amp;nbsp;</td><td>BP310100</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G-7021</td><td></td></tr><tr><td>Insulin</td><td>Novo Nordisk</td><td>393153</td><td></td></tr><tr><td>EGTA</td><td>Amresco</td><td>0732-288</td><td></td></tr><tr><td>Protease, type XIV</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>MEM</td><td>Corning</td><td>15-010-CV</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>JRS</td><td>43613</td><td></td></tr><tr><td>Primocin</td><td>Invitrogen</td><td>NC9141851</td><td></td></tr><tr><td>Ethyl Carbamate</td><td>Alfa Aesar</td><td>AAA44804-18</td><td></td></tr><tr><td>215 micron mesh</td><td>Component supply</td><td>U-CMN-215-A</td><td></td></tr><tr><td>20 G blunt ended needle</td><td>Becton Dickinson</td><td>305183</td><td></td></tr><tr><td>20 G beveled needle</td><td>Becton Dickinson</td><td>305176</td><td></td></tr><tr><td>Lab tape</td><td>VWR</td><td>89097-990</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-0</td><td></td></tr><tr><td>Silk suture, 7-0</td><td>Teleflex</td><td>15B051000</td><td></td></tr><tr><td>Mouse anti-alpha-actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG1 antibody</td><td>Thermo Fisher</td><td>A21121</td><td></td></tr></tbody></table>

isolation, culture and transduction of adult mouse cardiomyocytes

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.

A wild type adult ICR (CD1) mouse heart typically yields 500,000 to 1 million cardiomyocytes from a successful isolation. Immediately after isolation, the cells maintain a mostly rod-shaped appearance (Figure 3A) with intact sarcomeres and can be used for functional studies involving cardiomyocyte contractility. A high percentage of rod-shaped cardiomyocytes (above 90%) is an indication of effective perfusion and digestion. Viable cardiomyocytes will be large (~ 100 - 200...

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 ...

The overall goal of this procedure is to isolate viable primary adult mouse cardiomyocytes for culture and adenoviral vector transduction. This method will answer some the key question in the field of cardiovascular research. In particular, we can adjust what prevents adult mammalian cardiomyocytes from cell cycle entry and completion.The main advantage of this technique is that a high yield of viable adult mouse cardiomyocytes can be obtained for long term culture. Visual demonstration of this method is critical, as the heart extraction and cannulation procedures are difficult, but must be performed quickly and efficiently. To extract the heart, begin by treating an adult mouse with five international units of heparin per gram of body weight by intraperitoneal injection.Wait 20 minutes to allow the heparin to circulate to the heart. Then, after administering anesthesia, confirm a lack of response to toe pinch and corneal reflexes and tape the animal's limbs to a surgery platform. Sterilize the incision area, with liberal application of 70 percent ethynol, and then pat the hair dry with a lab wipe.Next, use forceps to lift the skin just below the sternum and make a lateral incision through the skin and peritoneum. Then, using hemostats, gently lift the rib cage via the sternum and use small dissection scissors to extend the incision in a V shape toward the axially. Cut through the first rib and puncture the diaphragm on each side, keeping the rib cage lifted with the hemostats.Using small Iris scissors, completely transect the diaphragm. Then use the hemostats to retract the ribcage rostrally and quickly but carefully extend the incision through the ribcage toward the axially on both sides. Retract the middle section of the ribcage fully, laying down the attached hemostats to hold the tissue in place, and remove the pericardium with fine forceps.Next, use curved forceps to gently lift the heart and transect the attached veins and arteries. Wash the heart in ice cold perfusion buffer, then transfer the heart onto a small piece of 215 micron mesh. Add ice cold perfusion buffer to slow myocardial contractions, and place the dish under a dissection microscope.Using fine iris scissors, trim any access non-cardiac tissue. Then transect the aorta near the brachiocephalic artery, taking care not to let bubbles or tissue debris enter the opening. To cannulate the heart, fill the Petri dish with ice cold perfusion buffer until the solution rises above the opening of the 18 gauge cannula.Then, keeping the tip of the cannula and aorta below the surface of the buffer, sheath the aorta onto the cannula until the needle tip is just distal to the aortic valve. Slide a pre-tied 7-0 silk suture down the shaft of the cannulation needle until the suture is just above the needle tip and tighten the knot with fine forceps. Place a second suture just above the first.Then slowly depress the syringe until 0.5 milliliters of perfusion buffer has been injected through the coronary vasculature. After removing the syringe, attach the cannula to the perfusion outlet port. At the end of the perfusion, use forceps to remove the heart from the cannula, and place the heart in an empty Petri dish.Using fine Iris scissors, quickly trim the extra ventricular tissue and wash the ventricles in a new Petri dish containing perfusion buffer. After washing, transfer the ventricles into a dry Petri dish and place in a sterile hood. Collect 7.5 milliliters of digestion buffer from the heat jacket of the perfusion system, and add 2.8 microliters of one molar calcium chloride.Mix the solution by inversion. And then add two to three milliliters of the calcium chloride supplemented digestion buffer to the ventricles. Using fine forceps, quickly tritcherate the ventricular tissue until most of the pieces are smaller than one cubic millimeter, and add the remaining calcium chloride supplemented digestion buffer to the dissociated tissues.Mix the fragments by gentle agitation. And incubate them at 37 degrees Celsius and five percent carbon dioxide with continued agitation every two minutes until a sufficient number of cardiomyocytes have been released. Then return the dish to the laminar flow hood and use a transfer pipette to gently dissociate the tissue further.Wearing sterile gloves, fold the 215 micron mesh into a cone and hold it over a 50 milliliter conical tube. Pipette the tissue slurry onto the mesh, and allow the filtrate to drain into the tube. Followed by a wash with 7.5 milliliters of pre-warmed stop buffer.Transfer the cell suspension to a 15 milliliter conical and allow the cells to settle by gravity for 15 minutes. Then use a new transfer pipette to carefully remove the supernatant until only 50 to 100 microliters of solution remains above the cells. Add 10 milliliters of pre-warmed equilibrated culture medium to the cells, and mix by gentle inversion.Finally, after another 15 minute room temperature incubation, use a new transfer pipette to remove the supernatant from the settled cells, until only 50 to 100 microliters of medium remain. Immediately after isolation, the cardiomyocytes maintain a mostly rod-shaped appearance with intact sarcomeres and a sharp outer membrane under bright field illumination. Immunostaining for cardiomyocyte-specific sarcomeric markers such as alpha actinin reveals a distinct sarcomeric banding pattern.After several days in culture, the cardiomyocytes assume a rounded morphology. Within one to two weeks, the live cardiomyocytes attach to the substrate and begin spreading. Transduction with adenovirus, as observed in these GFP positive cardiomyocytes, can be used to achieve a strong gene expression within 48 to 72 hours.Once mastered, this technique can be completed in four hours if it is performed properly. While attempting this procedure, it is important to remember to achieve a secure cannulation that is free of air bubbles in order to efficiently profuse the coronary vasculature. Following this procedure, other methods such as immunostaining or live imaging can be performed in order to answer questions such as why are adult mammalian cardiomyocytes resistant to cell division?Don't forget that working with adenovirus can be extremely hazardous, so appropriate precautions must be followed, such as working in a class two biosafety cabinet.

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Research

JoVE Journal

Biology

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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

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

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Isolation and Cultivation of Adult Rat Cardiomyocytes

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

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

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.

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Single cell expression profiling allows the detailed gene expression analysis of individual cells.  We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Learn to isolate and culture adult mouse cardiomyocytes with high yield and viability for cardiovascular research. This method enables adenoviral transduction to study cell cycle regulation in cardiac cells.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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