Development of new vessels is biological process called angiogenesis. It occurs during physiological processes such as embryogenesis , wound healing , female reproductive cycle  and bone formation . This process is also observed in many pathological conditions like cancerogenesis , diabetic complications , psoriasis  and rheumatoid arthritis .
Angiogenesis is a multistage process. It can be divided into few steps : 1. Activation of endothelial cells by different growth factors, 2. Extracellular proteinases destroyed the capillary wall, 3. Branch point are formed in the vessel wall, 4. Endothelial cells migrated into the extracellular matrix in the direction to angiogenic stimulus, 5. Endothelial cells form new tubules with a central lumen, 6. Anastomosis which means formation of branched network .
Matrigel tube formation assay is one of the most commonly used test to study angiogenesis. Matrigel matrix is reconstituted basement membrane specimen, which is extracted from tumors enriched in extracellular matrix proteins (e.g. Engelbreth-Holm-Swarm mouse sarcoma- EHS). It consists of approximately 60% laminin, 30% collagen IV and 8% entactin. It also contains many growth factors (e.g. TGF-β, EGF, bFGF, NGF, PDGF and IGF-1) and heparin sulfate proteoglycan. All these substances are present in EHS tumor. In the temperature between 22-37°C matrigel forms matrix gel, because it is enough energy to form chains between collagen IV, laminin and heparin. At 4°C matrigel becomes liquid solution because of not enough amount of energy to form these structures and connections. For studies about gene expression and explaining the role of growth factors scientists might use Growth Factors Reduced (GFR) type of Matrigel . The matrigel might cause differentiation of a variety of cell types like endothelial cells (e.g. HUVEC and TIME cell lines), hepatocytes (e.g. Huh-7 cell line) and mammary epithelial cells (e.g. MCF-7 cell line).
These assay might be applied for analysis of many substances, like small molecules and drugs, which can be useful as potential angiogenesis inhibitors in vascular therapies. It is performed in three basic steps: cell seeding, tube formation and angiogenesis observation. In the first step cells are seeded into cell culture plates (usually 48-wells) coated with matrigel (50-100µl per well). Cell density should be validated for all cell lines separately. After 24 hours cells form new tubes and cells are observed under transmitted-light microscope on low magnification. There are many commercially available kits for fluorescent staining of resulting vessels.
This assay is fast (24 hours from beginning to obtain results), easy to set up, and reproducible. The main difficult of this test is interpretation of the results. Software proposed by Dicella is easy to use, rapid and our photos do not require any additional processing (e.g. formation of binary system). This program uses different algorithms for the skeletonization of the created blood vessels. After analysis of our results, we can obtain information about branches, segments, pieces, meshes, nodes and junction. Results are in pixels (unit).
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