Angiogenesis, which denotes the process of developing new blood vessels from preexisting ones, is a vital event for embryonic development, organ growth and wound healing. Newly created blood vessels are responsible for supplying oxygen and nutrients to different tissues, removing waste products and promoting immune surveillance.
Among the most specific and well-defined in vitro tests for angiogenesis is the assessment of the propensity of endothelial cells to form 3D structures (also known as tube formation). Regardless of their origin, endothelial cells seem able to spontaneously form tubules, if they are provided with sufficient time in vitro to lay down adequate extracellular matrix components.
Principles of the tube formation assay
The tube formation assay represents a simple, expeditious and quantitative method for the determination of genes or different pathways that play a role in angiogenesis. Initially described in 1988, the fundamental principle of this assay is that endothelial cells preserve the capability to divide and rapidly migrate in response to certain angiogenic signals.
After discovering that Matrigel (which is a matrix-rich product derived from Engelbreth–Holm–Swarm tumor cells with laminin as a primary component) can elicit the formation of endothelial cell tube within 24 hours, these assays have attained a noteworthy place in our repertoire of angiogenesis measurement.
Culturing endothelial cells on a gel of basement membrane extract (BME) is a way to induce their differentiation and tube-like structures formation. Tubes that develop contain a lumen encircled by endothelial cells that are joined together via junctional complexes. Depending on the quantity and type of stimuli, tube formation can occur swiftly in this assay, with most tubes showing within 2 to 6 hours.
Different types of endothelial cells can be utilized for the purposes of this assay – including both primary and immortalized cell lines. Depending on the choice, a cell line is used and the use of transformed or non-transformed endothelial cells, optimization is often needed to identify the ideal time required for adequate tube formation.
Obtaining representative results
The choice of a right cell type for tube formation assay is pivotal for obtaining reliable results. Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) are standardized cells that are available from company Thermo Fisher Scientific. Some tumor cell lines (such as breast cancer MDA-MB-435 or melanoma B16F1 cells) are also able to form vascular tubules.
Matrigel matrix coating should be optimized by manufacturing process in order to generate flat surface and eliminate meniscus. Such Matrigel is available from BD Biosciences, which is also screened for its ability to promote efficient tube formation.
Cell count has a substantial impact on tube formation, as with more cells the tube network that can be obtained becomes more extensive. However, cell seeding density should also be taken into account, because high concentration can result in cell clumping or monolayer formation, thus any variations between treatment groups can become masked. Optimal seeding density is approximately 5 thousand cells per cm2.
Therefore standardized systems can significantly aid in obtaining best results. For example, Cultrex Angiogenesis System HT Tube Formation Kit, available from AMSBIO, allows easy detection of both inducers and inhibitors of cell tube formation, and also suits 96-well format assays.
Images can be documented via phase contrast microscopy by employing objectives between 4 and 20 times magnification. Nevertheless, if fluorescent images are required, a cell-permeant dye calcein AM can be added and the endothelial tube network visualized by using either fluorescence or confocal microscopy. Such stained HUVEC cells can be also detected using confocal collapsed stack imaging available from BD Biosciences.
In conclusion, the advantages of the tube formation assay are easy and short culture period, as well as possibility of quantification and high-throughput analysis. On the other hand, main disadvantage is a large variation of tube-forming ability among various lots of endothelial cells and matrices. Finally, all results should be confirmed in vivo.