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Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Published: August 28th, 2016



1Cardiovascular Research Institute, 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.

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.

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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 .......

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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.......

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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.......

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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.


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

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