Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Summary

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Abstract

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1 coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

Introduction

Angiogenesis, neurogenesis and neuronal migration are critical events in central nervous system (CNS) development, repair and regeneration. Several elegant studies have shown that endothelial cells stimulate neuronal proliferation and vice versa through release of soluble factors and by direct contact. We found it curious that in majority of these studies^1-3, while neuronal progenitors/neural stem cells are isolated from the embryonic brain, they are cocultured with endothelial cells from the adult brain, other adult tissue sources, or with endothelial cell lines. This might in part be due to the technical difficulties associated with isolating and culturin....

Protocol

1. Preparation of Reagents and Solutions

1. Coating of 35 mm culture dish: Collagen Type 1 solution is supplied as an aqueous solution in 20 mM acetic acid (~100 mg protein/vial). Dilute an appropriate volume of collagen solution to a working concentration of 0.01% using sterile tissue culture grade water. Coat dishes with 1 ml of collagen solution for 3-4 hr at room temperature (RT) or 37 °C, or overnight at 2-8 °C. Remove excess solution from the coated dish and allow it to dry overnight. Rinse the dish with tissue culture grade water or PBS before adding media. Coated dishes can be stored at 4 °C for one month.
2. Prepare complete Dul....

Results

The phenotypic characterization of PVECs from day 1-12 is shown by phase-contrast light microscopy (Figure 2). The cells attached to the dish on day 1 show morphology characteristic of cell division (Figure 2A). Between 5-8 days, PVECs transition from cobble stone to spindle shaped morphology typical for endothelial cells and more akin to its in vivo state (Figures 2B and 2C). By day 12 the PVEC culture achieves full confluence (Figure 2.......

Discussion

PVEC's are more physiologically relevant than adult brain endothelial cells and ECs from other tissue sources for studies focusing on neurovascular interactions and also have therapeutic potential. For PVEC preparation, it is critical beginning with dissection to work fast to achieve a good viability since dead cells may bind nonspecifically to CD31 MicroBeads. In addition, if single-cell suspension is not achieved prior to the magnetic labeling step, this will result in troubleshooting since cell clumps will clog th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DNase I	Sigma	D-4527
Collagen, Type 1 solution from rat tail	Sigma	C3867
DPBS	Quality Biologicals	114057-131
EDTA	Fisher Scientific	M4055
BSA	Sigma	A2058
MS column	Miltenyi Biotech	130-042-201
CD31 microbeads	Miltenyi Biotech	130-097-418
MACS separator	Miltenyi Biotech	130-042-102
MACS multi stand	Miltenyi Biotech	130-042-303
Cell strainer 70 µm	BD Bioscience	352350
Antibiotic and antimycotic solution	Sigma	A5955
FBS	Sigma	F4135
Collagenase/Dispase	Roche	10269638001
DMEM	Lonza	12-604F
35 mm Culture dish	BD Bioscience	353001
15 ml Falcon tube	BD Bioscience	352097
50 ml Falcon tube	BD Bioscience	352098
ECCM kit	BD Bioscience	355054	Kit Includes Endothelial cell growth supplement, EGF, and Soybean Trypsin Inhibitor
Endothelial Cell Growth Supplement (ECGS)	BD Bioscience	354006
RBECGM	Cell Applications	R819-500
DMEM F12	Life Technologies	10565-018
GlutaMAX	Life Technologies	305050-061
Tissue culture grade water	Life Technologies	15230162
0.25% Trypsin	Life Technologies	15050
Soybean trypsin inhibitor	BD Bioscience	5425
Matrigel	BD Bioscience	354234
Qtracker 655 Cell Labeling Kit	Life Technologies	Q25021
CellLight Plasma Membrane-RFP, BacMam 2.0	Life Technologies	C10608
Biotinylated Isolectin B4 antibody	Sigma	L2140
Anti-Von Willebrand factor	Sigma	F3520
Anti-CD31/PECAM-1	BD Pharmingen	550274
VECTASHIELD Hardset Mounting Media with DAPI	Vector Laboratories	H-1500
Ketamine	Butler Schein Animal Health Supply	44028
Xylazine	Lloyd Laboratories	1009
Stereomicroscope	Motic	SMZ-168
Hemocytometer	Fisher Scientific	267110
Inverted microscope	Olympus CK-40	CK-40
Fluorescent microscope	Olympus FSX-100	FSX-100
Fine forceps	Roboz Surgical Instrument	7 inox
Fine microtip scissors	Roboz Surgical Instrument	RS5611