Isolation of Mouse Coronary Endothelial Cells

Summary

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Abstract

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 10⁴ cells per one heart with over 90% purity.

Introduction

Mouse models of various cardiovascular diseases and metabolic disorders bear physiological and molecular changes which are similar to those found in patients. Furthermore, genetic alteration of mice is a powerful tool that allows us to investigate the pathogenic role of specific genes in the development and progression of diseases. Nowadays, cell-type-specific gene-knock-in or -out mice are easily generated in many laboratories and the measurement of mRNA and protein levels in specific cell types is the first step in determining whether the mice were truly genetically modified.

Endothelium is a thin single layer of endothelial cells (ECs) t....

Protocol

The research on mice was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arizona and was performed according to the National Institute of Health (NIH) guidelines.

NOTE: Sections 1, 2 and 3 should be carried out the day before the experiment as setup.

1. Preparing Solutions

1. CD31 buffer (50 ml)
   1. Add 50 ml 1× Phosphate Buffer Saline (PBS) to a 50 ml tube. Weigh 0.05 g Bovine Serum Albumin (BSA) and 0.037 g Ethylenediaminetetraacetic acid (EDTA) and add to the 50 ml tube and put the tube to rotate for about 1 h until powder dissolves. Adjust pH to 7.4, filter through a 0.2 µm filt....

Results

The successful isolation of coronary endothelial cells from a mouse heart typically yields around 10⁴ cells with over 90% purity with a cobblestone shape. For a pure population of endothelial cells, caution should be taken throughout the protocol to ensure a sterile environment free from any contamination. The appearance of elongated cells or enlarged cells within the population indicates contamination with other cell types, smooth muscle cells and fibroblasts. Endothelial cell.......

Discussion

The most widely used endothelial cells in research are human umbilical vein endothelial cells (HUVECs) because of their convenience and ease of culturing. However, a lot of research requires the availability of a physiological model attained by the isolation of endothelial cells from other specific organs¹⁰. Endothelial cells make up a very minor population of cells in the heart tissue; their isolation and culturing can prove to be very difficult.

There are three critical steps with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Sigma	A3311-10G
EDTA	Fisher	BP120-500
HBSS	Fisher	SH3026801
Hepes	Sigma-Aldrich	H4034-500G
Sodium Bicarbonate	Sigma	S5761
D-(+)-Glucose	Sigma	G7021-1KG
DynaBeads pan mouse IgG beads	Thermo Fisher Scientific	11035
Rat anti-mouse CD31 Antibody	BD Biosciences	550274
IFCS	Fisher	SH30072.04
M199	Fisher	MT10060-CV
Dispase (Neutral Protease)	Worthington Biochemical Corp./Fisher	LS02109
Collagenase Type II	Worthington Biochemical Corp./Fisher	LS004176
Glycerol	Fisher	BP381-1
Igepal	Sigma	I3021-50 ml
Phosphatase inhibitor cocktail	Sigma	P0044-1ml
Protease inhibitor cocktail	Sigma	P8340-1 ml
Heparin	Sigma	H3149-100KU
FBS	Fisher/Mediatech	MT35010CV
D-Valine	Sigma	V1255-5G
EGS	Corning	356006
Penicillin/Streptomycin	Fisher/Mediatech	MT-30-002-CI