Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Summary

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Abstract

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

Introduction

The vascular endothelium is not only a barrier layer that separates blood and tissue, it is considered a vast endocrine gland that stretches over the entire vascular tree with a surface area of 400 square meters¹. The well-being of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries, etc. ^2-4. To date, the specific cellular and molecular mechanisms involved in these disease settings are not well understood due to the diffused anatomic nature of endothelium.

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Protocol

1. Isolation of Aorta from Mice

All the procedures described here were approved by the Institutional Animal Care and Use Committee of Wayne State University.

1. Put the mouse into the induction chamber of the anesthesia machine. Set the isoflurane flow to 4% in conjunction with 25% fresh air and 75% O₂. Anesthetize the mouse by inhalation of isoflurane (4% for induction, ± 1.0% for maintenance) in conjunction with 25% fresh air and 75% O₂ via induction chamber followed by a dedicated nose cone. NOTE: Anesthesia with isoflurane can be delivered in 75% oxygen/25% air or in 100% oxygen. 

Results

Endothelial Cell Sprouting

Spontaneous endothelial cell sprouting started from a mouse aorta segment. The mouse aorta segment was allowed to grow on a growth factor-reduced matrix then in endothelial cell growth medium for 4 days. The endothelial cell sprouting usually appears in 2 - 4 days. Photomicrographs were taken on day 4 (Figure 1). As shown in the pictures, numerous endothelial cells migrate away from the segment. The newly formed sprouts continue to e.......

Discussion

This study demonstrates a simple method to isolate and culture endothelial cells from a mouse aorta without any special equipment. The immunofluorescence staining indicated that the majority of cells were endothelial cells after the second passage. It is suggested that cells at second to third passage are suitable for in vitro and in vivo experiments to study endothelial biology.

The Key Notes from the Present Protocol

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4- or 6-week-old mice	Jackson Laboratory	#000664
Sterile 1x phosphate-buffered saline (PBS)	Gibco	#10010-023
Sterile 1x PBS containing 1,000 U/mL of heparin	Sigma Aldrich	H3149
Endothelial cell growth medium (Dulbeccos’ Modified Eagle’s Medium [DMEM] with 25 mM HEPES [Gibco, #12320-032], supplemented with 100 μg/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/mL heparin [Sigma Aldrich, H3149], 10,000 U/mL penicillin and 10 mg/mL streptomycin [Gibco, #15140-122])
Growth factor reduced matrix	BD Biosciences	356231
Neutral proteinase (Dispase, 1 U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034 g/L, Sigma, #1255), in Dulbeccos’ Modified Eagle’s Medium, low glucose (Gibco, #12320-032)
1,1'-dioctadecyl-3,3,3',3'- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)	Life Technologies	L3484
FITC-labeled Ulex europaeus agglutinin (Ulex-Lectin)	Sigma	L9006
Anti-mouse CD31-FITC conjugated antibody	BD Biosciences	553372
Anti-mouse vascular endothelial growth factor receptor 2 antibody	Cell Signaling	9698
Anti-mouse endothelial nitric oxide synthase	Abcam	ab5589
Anti-mouse vascular endothelium-cadherin	Abcam	33168
Anti-mouse calponin	Abcam	700
FITC-conjugated anti-rabbit IgG	Sigma	F6005
1 mL syringe fitted with 25-G needle	Fisher Scientific	50-900-04222
100 mm Peri dishes	Fisher Scientific	07-202-516
Six-well cell culture plates	Fisher Scientific	08-772-1B
T12.5 culture flask	Fisher Scientific	50-202-076
Scissors, forceps, microdissection scissors and forceps, Scalpel blade	Fine Science Tools, Inc.
Anesthesia machine with isoflurane	Webster Veterianary Supply	07-806-3204
heating lamp
Centrifuge machine
Inverted phase-contrast microscope
inverted fluorescence microscope