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the Matrigel technique

Recently attempted to adapt the Matrigel technique, to circumvent some of the problems associated with the assay, namely the large quantity of expensive Matrigel used per test, the inability to quantify a whole well with ease, and the subjective choice of field of view, with the associated bias and the time-consuming nature of analysis. This was achieved by scaling down the assay for use in 384-well and 1536-well high-density formats where the endothelial cells yielded highly reproducible tubule formation per well. A computer-assisted integrated platform for capturing and processing images of complete wells was then used. This system evaluated the total number of nodes, connected and unconnected tubes as well as their lengths, and was tested in a double-blind experiment, which validated the results . This procedure has eliminated some of the problems of the Matrigel assays with respect to reproducibility and to the difficulties in analysing the tubule formation seen in a total well accurately and has resulted in an easy computerized system for analysing tubule formation. However, the lack of a lumen and the homogeneous nature of the tubules remain as limitations to this assay protocol.

It is also possible to use Matrigel and, more commonly, fibrin clots to represent a 3D angiogenesis system. In this assay, the endothelial cells are sandwiched between layers of matrix (either fibrin or Matrigel) and then allowed to form tubules over an extended period of time. Initially, endothelial cells form tubules in the horizontal plane, but over a period of 12 or more days, the endothelial tubes begin to branch upwards and penetrate the gel to form a 3D network of tubules.Although these assays more closely mimic the in vivo situation, in which endothelial cells do not just form capillaries in two dimensions, quantification of cell behaviour in three dimensions, which is notoriously difficult to analyse, remains a challenge. The analysis involves taking pictures at different heights (e.g. every 50–100 µm) in the gel from the bottom to the top. The length (both width, for those in the horizontal plane, and height, for those in the vertical plane) and largest diameter of each vessel are measured.The microvessel density can also be calculated using the Chalkley grid method where an eyepiece-mounted, 25-point Chalkley array graticule is used to assess the vascular density, by counting the number of points on the Chalkley grid coinciding with tubules at different heights through the gel (adapted fromThe limitations are obvious with only a proportion of tubules and the area or volume of the gel being analysed.