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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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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