Figure 5
Angiogenesis assays. Angiogenesis is a multi-step process, Different types of in vitro, in vivo or ex vivo bioassays have been designed to mimic the various steps of angiogenesis. (A) In vivo Matrigel Plug assay: liquid form Matrigel (500 μl) containing growth factor (e.g. bFGF) and/or ginsenoside is injected into the abdominal region of C57/BL mice subcutaneously. The Matrigel will solidify at 37°C and form a solid 'plug'. After 5 days of incubation, the mice are sacrificed and in vivo angiogenesis including endothelial cell invasion, migration and formation of neovessels can be examined [124]. (B) Ex vivo rat aortic ring sprouting assay: rat aortic fragments one millimeter in length are embedded in Matrigel and cultured in the presence of growth factors (e.g. endothelial cell growth supplements – ECGS) and/or ginsenosides. The extent of endothelial sprouting from the aortic fragment can clearly indicate the angiogenic properties of ginsenosides [124]. (C) In vivo sponge implantation assay: a sterile polyether polyurethane sponge (170 mm3) containing ginsenosides is inserted into the abdominal region of Balb/c mice. After incubation for 15 days, the animals are euthanized and neovascularization is examined as indicated by the growth of vessels in the granuloma tissue [129]. (D) In vitro tube formation assay: endothelial cells are seeded on the Matrigel and subsequently incubated in medium containing growth factors (e.g. VEGF) and/or ginsenosides. Endothelial cells will rearrange and alight into a tubular structure. Angiogenic properties of ginsenosides can be reflected from the number of tubes, branching points or tube area.