The process by which new blood vessels are formed from pre-existing vasculature is called angiogenesis.
Angiogenesis is important in embryonic development and reproduction. It is also essential in wound healing. When insufficient new blood vessels are formed chronic wounds can occur.
Pathological angiogenesis can also occur when too many new blood vessels are formed. This is key to tumor growth and metastasis of cancers. Excessive angiogenesis is also thought to be involved in diabetic retinopathy, atherosclerosis and inflammatory diseases such as rheumatoid arthritis.
In vitro and in vivo angiogenesis assays
A variety of in vitro assays can be useful in determining the progress and/or extent of angiogenesis. These include:-
- Tube formation assays
- Proliferation of endothelial cells
- Migration of endothelial cells in response to wound healing
- Cell invasion and migration
In vivo angiogenesis assays may also be useful. These include:-
- Directed in vivo angiogenesis assay (DIVAA)
- Rat aortic ring
- Chick chorioallantoic membrane (CAM)
- Corneal micropocket
- Basement membrane subcutaneous plug
Tube formation assays
Tube formation assays assess the differentiation of endothelial cells and the formation of tube-like structures on extracellular matrices such as Basement Membrane Extract (BME). Basement Membrane Extract (BME) is a matrix-rich product prepared from Engelbreth–Holm–Swarm (EHS) tumor cells whose primary component is laminin. It can lead to endothelial cell tube formation within 24 hours.
These assays are useful when screening a drug for angiogenic activity, or determining whether a factor has an effect on angiogenesis.
Studies have shown that endothelial cells of all origins are able to form tubules spontaneously if they are allowed to lay down in appropriate extracellular matrix components.
If collagen or fibrin clots are used to coat plastic culture dishes, tube formation may be enhanced.
Optimal conditions for tube formation assays
Cell health is critical for successful results in tube formation assays. For this the cells should be allowed to recover after freezing and they should be plated 24 hours prior to assay. Primary cell lines beyond passage10-12 should not be used and cells should not grow to more than 80% confluent.
High performance and quality assured CELLBANKER and STEM-CELLBANKER cryopreservation solutions are available from AMSBIO to ensure stable long-term storage of stem cells.
The extracellular matrix should be chosen according to experimental design. For example, normal BME promotes efficient tube formation by endothelial cells in the basal medium without additional angiogenic factors.
Reduced Growth Factors (RGF) BME on the other hand allows for a low level of tube formation in the basal medium.
Further optimal seeding density is about 5,000 cells per cm2.
Duration of the assay depends on cell type. For example, primary endothelial cells take 6-20 hours to develop tube like network, while immortalized cells will form tubes in 2-3 hours.
Factors that affect tube formation assays
There are several factors that can affect tube formation assays. These include:-
- Extracellular matrix microenvironment, including the type, thickness and density of matrix proteins used
- Cell health, number of passages of the cell culture and confluency
- Cell seeding density
- Induction/inhibiting factors of angiogenesis
- Duration of the assay in hours
Cultrex ® In Vitro Angiogenesis Assay Kit
The Cultrex® In Vitro Angiogenesis Assay Kit, available from AMSBIO, is a cost-effective method for investigating inhibitors and inducers of angiogenesis in a 96-well plate format.
This Tube Formation Assay Kit utilizes a reduced growth factor BME that can detect low levels of tube formation in absence of the angiogenic factors and also detect high levels of tube formation in presence of angiogenesis stimulants using human umbilical vascular endothelial cells (HUVEC).
Endothelial Progenitor cells are also available from AMSBIO separately.
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