Name: isolation, culture and transduction of adult mouse cardiomyocytes
Uploaded: 2016-08-28T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

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 ...

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.

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			<td height="20" style="height:20px;">Heated water jacket</td>
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			<td style="width:99px;">11251-20</td>
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			<td height="21" style="height:21px;">Small dissection scissors</td>
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			<td style="width:99px;">470128034</td>
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			<td height="21" style="height:21px;width:295px;">Extra fine bonn scissors</td>
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			<td height="21" style="height:21px;width:295px;">Fine spring scissors</td>
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			<td height="21" style="height:21px;">MgSO4-7H2O</td>
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			<td height="21" style="height:21px;">Taurine</td>
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			<td style="width:99px;">86329</td>
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			<td style="width:99px;">B0753</td>
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			<td style="width:99px;">0732-288</td>
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			<td height="21" style="height:21px;width:295px;">Protease, type XIV</td>
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			<td style="width:99px;">P5147</td>
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			<td style="width:99px;">AAA44804-18</td>
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			<td height="21" style="height:21px;width:295px;">20 G blunt ended needle</td>
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			<td height="21" style="height:21px;width:295px;">20 G beveled needle</td>
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			<td style="width:99px;">305176</td>
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			<td height="21" style="height:21px;width:295px;">Lab tape</td>
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			<td style="width:148px;">3M</td>
			<td style="width:99px;">1527-0</td>
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			<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>
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		<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>
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		<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>
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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 ...

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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