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 border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Equipment</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated water jacket</td>
			<td>Radnoti</td>
			<td>158831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Circulating heated water bath, Isotemp</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:99px;">3013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Laboratory pump</td>
			<td style="width:148px;">Watson-Marlow</td>
			<td style="width:99px;">323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hemostats</td>
			<td style="width:148px;">Exelta</td>
			<td style="width:99px;">63042-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 curved forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #5 fine forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small dissection scissors</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Extra fine bonn scissors</td>
			<td>FST</td>
			<td style="width:99px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fine spring scissors</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91500-09</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Materials</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NaCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">KCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25837</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">86329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butane dione monoxime (BDM)</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:148px;">Fisher&#160;</td>
			<td style="width:99px;">BP310100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">G-7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Insulin</td>
			<td style="width:148px;">Novo Nordisk</td>
			<td style="width:99px;">393153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EGTA</td>
			<td style="width:148px;">Amresco</td>
			<td style="width:99px;">0732-288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Protease, type XIV</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Collagenase II</td>
			<td style="width:148px;">Worthington</td>
			<td style="width:99px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:99px;">15-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">FBS, heat inactivated</td>
			<td style="width:148px;">JRS</td>
			<td style="width:99px;">43613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Primocin</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:99px;">NC9141851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethyl Carbamate</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">AAA44804-18</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">215 micron mesh</td>
			<td style="width:148px;">Component supply</td>
			<td style="width:99px;">U-CMN-215-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G blunt ended needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G beveled needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Lab tape</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">89097-990</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">Surgical tape</td>
			<td style="width:148px;">3M</td>
			<td style="width:99px;">1527-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Silk suture, 7-0</td>
			<td style="width:148px;">Teleflex</td>
			<td style="width:99px;">15B051000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mouse anti-alpha-actinin antibody</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">A7811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Alexa Fluor 488 goat anti-mouse IgG1 antibody</td>
			<td style="width:148px;">Thermo Fisher</td>
			<td style="width:99px;">A21121</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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 ...

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26.4K Views.  University of California, San Francisco. 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. 

Video: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

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.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

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.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

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.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

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.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

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.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

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

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.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...