Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Summary

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Abstract

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Introduction

Angiogenesis, the development of new blood vessels from preexisting vessels, is vital for a variety of processes including organ growth, embryonic development, and wound healing^1-3. Newly developed blood vessels, lined by endothelial cells, supply oxygen and nutrients to tissues, promote immune surveillance by hematopoietic cells and remove waste products^2,4. Angiogenesis is of key importance during embryonic and fetal development. However, this process remains dormant in the adult except during times of wound healing, skeletal growth, pregnancy or during the menstrual cycle^1-3.

Over the last two decades, key molecular mechanisms that regulate angiogenesis have begun to emerge. Angiogenesis is a tightly regulated event, balanced by pro and antiangiogenic signals including integrins, chemokines, angiopoietins, oxygen sensing agents, junctional molecules and endogenous inhibitors⁵. Once proangiogenic signals such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and epidermal growth factor (EGF) activate endothelial cell receptors the endothelial cells release proteases to degrade the basement membrane. The endothelial cells then proliferate and migrate, forming sprouts at a rate of several millimeters per day^6,7.

Angiogenesis is associated with various pathological conditions including cancer, psoriasis, diabetic retinopathy, arthritis, asthma, autoimmune disorders, infectious diseases, and atherosclerosis^8-10. Due to the importance of angiogenesis in various diseases, understanding the genes and pathways that regulate this process is critical to the design of better therapeutics.

The tube formation assay is a rapid and quantitative method for determining genes or pathways involved in angiogenesis. First described in 1988, the principles underlying this assay are that endothelial cells retain the ability to divide and migrate rapidly in response to angiogenic signals^11-13. Further, endothelial cells are induced to differentiate and form tube-like structures when cultured on a matrix of basement membrane extract (BME). These tubes contain a lumen surrounded by endothelial cells linked together through junctional complexes. Tube formation occurs quickly with most tubes forming in this assay within 2-6 hr depending on quantity and type of angiogenic stimuli.

Several types of endothelial cells can be used for this assay including both primary cells and immortalized cell lines^14,15. The cell line used for this article was mouse 3B-11, but the same methodology can be applied with other endothelial cell lines such as SVEC4-10 (mouse) or primary endothelial cells such as HUVEC (human) cells. Depending on which cell line is used and whether the endothelial cells are transformed or non-transformed, optimization will need to be conducted to identify the ideal time needed for proper tube formation.

Protocol

1. Collection of Conditioned Media to Test for Angiogenic Potential Grow primary or immortalized cells to be tested for angiogenic or anti-angiogenic potential in native or low serum media and collect the conditioned media. Alternatively, use conditioned media immediately, or aliquoted and stored at -80 °C for several months. Use nonconditioned native or low serum media as a negative control, and use non-conditioned complete growth media (10% FBS, or appropriate concentration) as a positive c…

Representative Results

Mouse 3B-11 endothelial cells were seeded on solidified reduced growth factor BME – in this assay, the product Matrigel was used – and followed over time. As shown in Figure 1, angiogenic factors secreted by either mouse keratinocytes or fibroblasts are capable of inducing tube formation over time. Endothelial cells migrate and begin to form small branches within 1-2 hr of plating. Maximum tube formation was reached by 4-6 hr using conditioned media previously obtained from keratinocytes. By 24 hr,…

Discussion

Angiogenesis is involved in both physiological and pathological processes. Studying mechanisms involved in angiogenesis requires the use of assays that recapitulate the major steps in angiogenesis. The endothelial cell tube formation assay offers several advantages over other assays. It is easy to set-up, is relatively inexpensive, produces tubules within hours, and is quantifiable. Further, it can be completed in 24- or 96-well plates, and thus can be used for high throughput screening to identify factors that stimulate…

Materials

Item	Manufacturer	Catalog #
Costar 24-Well Tissue Culture-Treated Plate	Corning	3524
BD Matrigel Basement membrane matrix, Growth factor reduced	BD Biosciences	354230
Gibco 0.05% Trypsin-EDTA	Life Technologies	25300-054
Gibco L-Glutamine 200mM (100X)	Life Technologies	25030-081
Gibco Pen Strep	Life Technologies	15140-122
Gibco DMEM (1X)	Life Technologies	11965-092
Gibco DPBS (1X)	Life Technologies	14190-144
Fetal Bovine Serum	Atlas Biologicals	F-0500-A
Calcein AM	Life Technologies	C3100MP
DMSO	ATCC	4-X