Question

What are Tube Formation Assay technique's limitation? Does it fully represent in vivo tissue? How can the technique be improved?

Answer 1

answer) One major problem is that, because there are different types of endothelial cells and support matrices, the results of the assay can vary greatly depending on which type of cell and matrix is used.

Endothelial cells are the most important tool for in vitro studies. Human umbilical vein endothelial cells (HUVEC) are the commonly used cells for in vitro angiogenesis assays However, as the process of angiogenesis involves the microvasculature instead of macrovasculature, these assays are far from the ideal conditions because of the difference in the lineage and may lead to inadequate or erroneous responses. One problem with endothelial cell assays is the phenotypic differences in endothelial cells and these differences are not always of the same kind. This difference can be observed clearly between the endothelial cells of large vessels and endothelial cells of microvascular origin. In addition, in vitro endothelial cells may exhibit altered characteristics when compared the resting conditions with flowing culture conditions as well as when put in comparison by means of attachment to different matrices. Therefore, even if in vitro analysis methods are quick, easily assessable and reproducible, these bind the study of the complex physiological communications which take place in vivo. Furthermore, the evaluations of the secondary effects of a compound (which are employed on non-endothelial cells that in turn produce chemicals which act on endothelial cells) are difficult to imitate. So, more importantly, in vitro angiogenesis assays should be followed up with one or more in vivo assays of angiogenesis.

The purpose of the present study was to improve the widely used tube formation assay system in three respects. First, we used human ECFCs, endothelial precursors, for the assay. Second, we utilized a real-time cell recorder (NanoEnTek, Seoul, Korea) to continuously monitor tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view as large as 2×2 stitched area from the 4× object lens. This advanced tube formation assay system successfully measured the time-dependent progression of tube formation.