Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

Summary

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Abstract

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Introduction

Rodent models have long been used as tools to broaden our understanding of cardiovascular physiology in health and disease.¹ Although these animal models allow us to understand the pathophysiology of a disease at the organ level and to analyze the pharmacokinetics and pharmacodynamics of various pharmacological agents used to treat cardiovascular diseases, understanding of the molecular mechanisms of cardiovascular disease development and the contribution of a particular cell type requires the use of in vitro cell culture models. For this purpose different immortalized cell lines from the cardiovascular system have been developed;

Protocol

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 1985) and was approved by the local ethics committee of the University of Giessen. Adult male Wistar rats weighing 200 - 250 g were used in this study.

1. Autoclaving

1. At least one day before the procedure is to be carried out, autoclave all surgical instruments, pipette tips, and Pasteur pipettes to be used in the isolation procedure at 121 °C for 30 min.

2. Preparation of Media and Solutions

NOTE: Solution....

Results

The isolation procedure results in a yield of 70 - 80% viable, rod-shaped, striated cardiomyocytes (Figures 2A and 2C) that can be used for planned experiments. In our laboratory cardiomyocytes are routinely used for analysis of Ca²⁺ signaling. Figure 5A shows intracellular calcium [Ca²⁺]_i oscillations in response to ischemia/reperfusion in cardiomyocytes loaded with Fura-AM (5 µM). Figure.......

Discussion

In this article, a reproducible protocol for the isolation and culture of cardiac myocytes, endothelial cells, and fibroblasts is described. This protocol describes the simultaneous and high quality isolation of the major cell types of cardiac tissue, i.e. cardiomyocytes, endothelial cells, and fibroblasts as opposed to just one cell type. A critical step in the isolation procedure is the proper digestion of the cardiac tissue. If the digestion is incomplete, a large number of myocytes may still be obtained but .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-vWF	Santa Cruz Biotech.	SC-14014
Calcium chloride	Merck	102378
Carnitin	Sigma-Aldrich	C0283
Collagenase type II	Worthington	LS004176
Creatin	Sigma-Aldrich	C0780
D-Glucose	Merck	108342
Dil-Ac-LDL	Thermo Scientific	L3484
EDTA Solution (0.2 M)	Biochrome AG	L2113
Embeding solution	Citiflour	AF1-25
Endothelial cell medium MV2	PromoCell	C-22022
Foetal calf serum (FCS)	Biochrome AG	S0615
Gentamicin	Serva Chemicals	47991
HEPES	Sigma-Aldrich	H0887
Isoflurane	Abbott	TU 061219
Laminin	Roche/Sigma	11243217001
M199 medium	Thermo Scientific	11150059
M199 medium (Powder)	Biochrome AG	T061
Magnesium sulphate	Sigma-Aldrich	63138
Mouse anti-rat CD31 antibody (TLD-3A12)	Thermo Scientific	MA1-81051
NaCl solution (0.9%), Sterile	B. Braun	30820080
Pan mouse IgG beads (Dynabeads)	Thermo Scientific	11041
Paraformaldehyde (PFA) 4% Solution	Santa Cruz Biotech.	sc-281692
Penicillin-Streptomycin	Thermo Scientific	15070-063
Phosphate buffer saline (PBS) 1x	PAN-Biotech	P04-36500
Plastic consumables	Greiner Bio-One
Potassium Chloride	Merck	4933
Potassium dihydrogen phosphate	Merck	7873
Sodium Chloride	Merck	6404
Sodium Hydroxide Solution (2 N)	Merck	109136
Sterile filtration system	Thermo Scientific	5660020
Taurine	Sigma-Aldrich	T8691
TO-PRO	Thermo Scientific	T3605
Trypsin-EDTA Solution (10x)	Sigma-Aldrich	T4174
Water, Sterile	B. Braun