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Biology
Isolement, culture et caractérisation fonctionnelle des adultes Cardiomyoctyes souris
Isolement, culture et caractérisation fonctionnelle des adultes Cardiomyoctyes souris
JoVE Journal
Biology
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JoVE Journal Biology
Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Isolement, culture et caractérisation fonctionnelle des adultes Cardiomyoctyes souris

Full Text
25,537 Views
12:49 min
September 24, 2013

DOI: 10.3791/50289-v

Evan Lee Graham1, Cristina Balla1,2, Hannabeth Franchino1, Yonathan Melman1, Federica del Monte*1, Saumya Das*1

1Cardiovascular Institute,Beth Israel Deaconess Medical Center, Harvard Medical School, 2Division of Cardiology,Sapienza University

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article describes a procedure for isolating adult mouse cardiomyocytes using a Langendorff perfusion system. The isolated cells are suitable for culture and transfection, allowing for gene expression manipulation and functional analysis.

Key Study Components

Area of Science

  • Cardiovascular biology
  • Cell culture techniques
  • Electrophysiology

Background

  • Isolation of cardiomyocytes is crucial for studying heart function.
  • Langendorff perfusion allows for effective cell isolation.
  • Understanding calcium handling in cardiomyocytes is essential for cardiac research.
  • Transfection techniques enable gene manipulation in isolated cells.

Purpose of Study

  • To isolate adult mouse cardiomyocytes for functional studies.
  • To visualize contraction and calcium handling in these cells.
  • To facilitate gene expression manipulation through viral transfection.

Methods Used

  • Langendorff perfusion system for heart isolation.
  • Enzyme digestion to prepare cardiomyocytes.
  • Cell culture techniques for maintaining isolated cells.
  • Patch clamp techniques for analyzing cell functionality.

Main Results

  • Isolated cardiomyocytes are Ca2+-tolerant and electrically quiescent.
  • Cells can be cultured and transfected with adeno- or lentiviruses.
  • Functional analysis reveals insights into contraction and calcium handling.
  • Results support the use of these cells for further cardiac research.

Conclusions

  • The Langendorff perfusion system is effective for isolating cardiomyocytes.
  • Isolated cells are suitable for various experimental manipulations.
  • Findings contribute to understanding cardiac cell function and gene expression.

Frequently Asked Questions

What is the Langendorff perfusion system?
It is a method used to isolate hearts from animals for research purposes, allowing for the study of cardiac function.
How are cardiomyocytes cultured after isolation?
The isolated cardiomyocytes are placed in a suitable culture medium and conditions that support their growth and functionality.
What techniques are used to analyze cardiomyocyte functionality?
Patch clamp techniques and MMSYS systems are used to assess contraction and calcium handling in the cells.
Can gene expression be manipulated in isolated cardiomyocytes?
Yes, the cells can be transfected with adeno- or lentiviruses to manipulate gene expression.
What are the main applications of isolated cardiomyocytes?
They are used for studying heart function, drug effects, and gene expression in cardiac research.
What is the significance of calcium handling in cardiomyocytes?
Calcium handling is critical for cardiac contraction and overall heart function, making it a key area of study.

Nous décrivons ici l'isolement de cardiomyoctyes de souris adultes en utilisant un système de perfusion de Langendorff. Les cellules résultantes sont des ions Ca2 + à tolérance de repos électriquement et peuvent être cultivées et transfectées avec l'adénovirus ou de lentivirus de manipuler l'expression génique. Leur fonctionnalité peut également être analysé en utilisant le système MMSYS et techniques de patch-clamp.

L’objectif global de cette procédure est d’isoler la culture et de visualiser la contraction et la manipulation du calcium dans les cardiomyocytes miroirs adultes. Pour ce faire, il suffit d’abord de retirer le cœur du muren et de le placer à température ambiante. PBS, le cœur peut être canulé dans la boîte comme le montre le dessin animé ou directement sur l’aiguille attachée au système de perfusion, comme dans le film.

La troisième étape consiste à laver et à digérer le cœur en pompant la perfusion, le tampon, puis le tampon enzymatique à travers le système. La dernière étape consiste à couper les ventricules dans un bol de tampon de transfert et à hacher doucement le tissu avec une pince. En fin de compte, les résultats peuvent montrer la contraction des cellules cardiomyocytaires individuelles et la manipulation du calcium par le biais de mesures de longueur de cellules entières et/ou de sarcomères et de ratio.

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Biologie cellulaire Numéro 79 médecine cardiologie biologie cellulaire anatomie physiologie souris canaux ioniques culture cellulaire primaire électrophysiologie cardiaque les cardiomyocytes de souris adultes l'isolement cellulaire IonOptix culture cellulaire transfection adénoviral patch clamp nanocapteurs fluorescents

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