A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

Summary

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

Abstract

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

Introduction

For decades, an essential idea in cardiac biology literature is the molecular mechanism of action. The mechanism of action must be established in order to publish reliable studies. A well-established strategy to determine molecular mechanism is isolated cardiomyocyte studies, which require high-quality cardiomyocytes for attaining trustable data. Cellular and molecular experiments performed on cardiomyocytes to determine mechanism of action are the gold standard for investigating contraction¹, electrophysiology², calcium (Ca²⁺) cycling³, myofilament Ca²⁺ sensitivity

Protocol

All procedures performed in this study were approved by the Institutional Animal Care and Use Committee at the Ohio State University in accordance with NIH guidelines.

1. Solution preparation

NOTE: Please see Table 2 for buffer concentrations.

1. Pre-isolation (up to 2 weeks in advance of myocyte isolation)
   1. Perfusion buffer base: Prepare 1 L of perfusion buffer base (NaCl, KCl, NaH₂PO₄, HEPES, Glucose, and BDM) in 1 L of ultrapure 18.2 MΩ·cm H₂O. Adjust to pH 7.4 prior to 0.22 µm sterile filtration. Store at 4 &....

Results

There are a few elements to examine when determining the success of an isolation. First, the cardiomyocytes must be rod-shaped with no membrane blebs, such as the cells isolated in Figure 1. A typical isolation will yield ~80% of the myocytes being rod-shaped. If the isolation yields anything less than 50% rod-shaped cells, then it is considered an unsuccessful isolation and cardiomyocytes are not used. Lastly, the cardiomyocytes should be quiescent. Spontaneously contraction myocytes demons.......

Discussion

The principal advantage of our Langendorff-free cardiomyocyte isolation technique is that it limits hypoxia and ischemic time by not requiring cannulation to a Langendorff apparatus. Alternatively to classical Langendorff techniques that take several minutes to remove, clean, and hang the heart, often resulting in ischemic damage to the myocyte, our method includes an in vivo clearing of blood via an ice-cold clearing solution. The ice-cold clearing buffer contains ethylenediaminetetraacetic acid (EDTA).......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 cc Bd Luer-Lok Syringe	Fisher Sci	14-827-52
10 mL Pyrex Low-Form Beaker	Cole-Palmer	UX-34502-01
100 mL polypropylene cap glass media storage bottle	DWK Life Sciences	UX-34523-00
14 mL Round-Bottom Polypropylene Test Tubes With Cap	Fisher Sci	14-959-11B
2,3-Butanedione Monoxime	Sigma	B0753	>98%
3 cc BD Luer-Lok Syringe	Fisher Sci	14-823-435
35 mm glass bottom dishes	MatTek Corporation	P35G-1.0-20-C
50 mL BD Syringe without Needle	Fisher Sci	13-689-8
50 mL Conical Centrifuge Tubes	Cole-Palmer	EW-22999-84
95% O2 5% CO2
AIMS Space Gel Heating Pad	Fisher Sci	14-370-223
BD PrecisionGlide 27 G X 1/2" Hypodermic Needles	Becton Dickinson	305109
Bovine Serum Albumin	Sigma	A3803	Heat shock fraction, lyophilized powder, essentially fatty acid free, >98%
Calcium Chloride dihydrate	Sigma	C7902	>99%
D-(+)-Glucose	Sigma	G7021	Suitable for cell culture, >99.5%
DMEM	Fisher Sci	11965092
EDTA	Fisher Sci	AAA1071336
Falcon 100 mm TC-treated Cell Culture Dish	Corning	353003
FBS	R&D Systems (Bio-techne)	S11195
Fisherbrand Isotemp Heated Immersion Circulators	Fisher Sci	13-874-432
Hartman Mosquito Hemostatic Forceps	World Precision Instruments	15921
Hausser Scientific Hy-Lite Counting Chamber Set	Fisher Sci	02-671-11
HEPES	Sigma	H4034	>99.5%
Labeling Tape	Fisher Sci	15-901-10R
Legato 100 Syringe Pump	kdScientific	788100
L-glutathione	Fisher Sci	ICN19467980
Liberase TH Research Grade	Sigma	5401135001	High thermolysin concentration
M199	Fisher Sci	MT10060CV
Magnesium Chloride	Invitrogen	AM9530G
Mouse Laminin	Corning	354232
Pen/Strep	Fisher Sci
Potassium Chloride	Sigma	P5405	>99%
Precision Digital Reciprocating Water Bath	ThermoFisher Scientific	TSCIR19
Sodium Bicarbonate	Sigma	S5761	Suitable for cell culture
Sodium Chloride	Sigma	S5886	>99%
Sodium phosphate monobasic	Sigma	S5011	>99%
Sterile Cell Strainer 70 µm	Fisher Sci	22-363-548
Student Fine Scissors	Fine Science Tools	91460-11
VWR Absorbent Underpads	Fisher Sci	NC9481815