Published: August 28th, 2016
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 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.
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.
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 .......
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.......
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 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.......
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.....
|Heated water jacket
|Circulating heated water bath, Isotemp
|Dumont #7 curved forceps
|Dumont #5 fine forceps
|Small dissection scissors
|Extra fine bonn scissors
|Fine spring scissors
|Butane dione monoxime (BDM)
|Protease, type XIV
|FBS, heat inactivated
|215 micron mesh
|20 G blunt ended needle
|20 G beveled needle
|Silk suture, 7-0
|Mouse anti-alpha-actinin antibody
|Alexa Fluor 488 goat anti-mouse IgG1 antibody
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