What are the in vitro angiogenesis assays?
Posted October 10, 2023
The in vitro angiogenesis assays include: endothelial cell proliferation assays, endothelial cell differentiation assays, endothelial cell migration assays, endothelial-mural cell co-culture assays, and ex vivo assays (organ culture).
- In sprouting angiogenesis, endothelial cells undergo proliferation and these assays are highly reproducible, simple, and produce precise data. However, endothelial cells also become senescent and proliferation assays need to be carried out soon following isolation. The simplest way of measuring the number of cells dividing is by determining the net cell number. In these assays, a specific amount of endothelial cells are seeded and after a period of time to allow proliferation to occur (typically 24 hours), the increase in the cell number is measured by using a coulter counter (an electronic particle counter), haemocytometer, or Vi-cell counter (which measures both cell viability and number). The more novel Vi-cell counter uses trypan blue exclusion to give total cell numbers as well as the percentage of dead cells. This is the most accurate method, but is also the most expensive of the 3.
- Another common technique for cell proliferation is MTT assay, in which a yellow tetrazolium salt is taken up by cells and cleaved by a dehydrogenase enzyme to produce purple formazan crystals. The crystals are then dissolved by the addition of DMSO and the solubilized formation is quantified using an ELISA plate reader. During angiogenesis, endothelial cells are stimulated to degrade the basement (after releasing proteases) and migrate to the perivascular stroma in response to angiogenesis growth factors (e.g. VEGF).
- Transfilter assays are the most commonly used assays to quantitatively analyze endothelial cell migration. These are 3D assays based on the migration of endothelial cells, placed on top of a filter with 8 micrometer diameter pores, and only allows active passage of the cells towards an attractant placed in the lower chamber. The filter may be coated with single ECM proteins like collagen or fibronectin, or complex matrices such as Matrigel to stimulate the in vivo microenvironment. The advantages of these assays is that they provide high sensitivity to small differences in concentration gradients and no proliferative effects. These assays also are used to distinguish between chemotaxis and chemokinesis. Assays which stimulate the formation of capillary-like tubules are seen as the marker of later stages of angiogenesis and are used to assay novel compounds for pro- or anti-angiogenic effects.
- The basic tubule formation for cell differentiation assay involves plating endothelial cells onto or into a layer of gel matrix (typically fibrin, Matrigel, or collagen), which stimulates the binding, migration and differentiation of these cells into tubule-like structures. Cell differentiation assays typically function in the presence of modulators of angiogenesis and tubule development is seen over a 4-24 hour period. They are recorded using a digital camera. The type of matrix utilized is important to consider as different matrices result in different rates of differentiation. For example, culturing endothelial cells on collagens I and III result in cell proliferation with only occasional tubule formation. In contrast, plating on collagen IV and V lead to extensive tubule formation with minimal proliferation.
- Endothelial-Mural cell co-culture assays are used after endothelial cells have coalesced to form tubules. Mural cells are recruited to maintain vessel quiescence and stability. A crucial part of the study of angiogenesis is the interaction between mural and endothelial cells. The most basic co-cultures involve seeding the cells simultaneously or allowing one cell to adhere type first and then seeding the second cell type on top. To analyze the interaction/influence of one cell type to the other’s behavior, it is necessary to label at least one population before seeding. Fluorescently labeled cells are particularly useful as they allow for the monitoring of cell activities (migration, tubule formation, proliferation) in real time. To analyze the effect of one cell type on the proliferation of the other, cells can be labeled in situ in the co-culture well with BrdU and immunostained with antibodies against BrdU. This is done to identify proliferation cells in conjunction with co-staining with cell type specific markers to induce the growth of each population to be quantified. These assays have been utilized to study effects of endothelial cells on mesenchymal cell differentiation, and analysis of gap junctions.
- Ex vivo assays have discs or sections of the specific tissue type that are cultured in a 3D matrix in vitro and are monitored for microvessel outgrowth over a period of 10-14 days. Quantification in these assays is achieved by measuring the length and number of microvessel outgrowths from primary explants. In specific, accurate quantification requires determining the number and length of branching microvessels, the number and spatial distribution of fibroblast-like cells, and the size and form of the original tissues. These can be counted by eye (quantity of vessels and branch points) or digitally (area, sprouting index). These assays are considered to induce the in vivo angiogenesis microenvironment because the system includes the surrounding non-endothelial cells (e.g. pericytes and smooth muscle cells) and a supporting matrix.