Angiogenic sprouting is the growth of new blood vessels from pre-existing vessels. This is orchestrated by cues from within the cellular microenvironment such as biochemical gradients and perfusion. The process of angiogenesis involves the formation of tip cells that invade the extracellular matrix, followed by the formation of stalk cells, phalanx cells, and eventually perfusable sprouts. The process of angiogenesis and the subsequent formation of microvasculature is crucial in studying the onset and progression of many diseases, including cancer, macular degeneration, Alzheimer’s disease, vascular fibrosis and inflammation. In addition, it also plays a role in kidney failure associated with diabetes.
Banner above: Confocal image of a blood vessel (top) having angiogenic sprouts through a collagen gel (centre).
Depiction of a 3D approach to study angiogenesis and microvasculature by culturing perfusable 3D angiogenic sprouts in the OrganoPlate® 3-lane, a membrane-free platform. A vessel of human endothelial cells is grown against an ECM gel, after which a cocktail of angiogenic factors is added to the other side of the gel. The resulting gradient of angiogenic factors induces the formation of endothelial sprouts through the ECM gel in 4 days. The platform’s compatibility with standard laboratory equipment and automation makes this perfused 3D angiogenesis model amenable to high-throughput screening.
The OrganoPlate®, a high-throughput microfluidic platform, was used to establish the angiogenesis model. The OrganoPlate® 3-lane comprises 40 tissue culture chips, each consisting of three channels (Figure 1a). An ECM gel was seeded in the middle channel of each chip and is patterned by phaseguides. Endothelial cells are grown in the top channel of each chip and form an endothelial vessel under perfusion by placing the OrganoPlate® on the OrganoFlow platform. Addition of a cocktail of angiogenic factors in the bottom channel induces the directed formation of angiogenic sprouts (figure 1b).
Analysis and quantification
Characterization of perfusable angiogenic sprouts. Cultures were stimulated with an angiogenic cocktail in the bottom channel for 0, 1, 2, 3, and 4 days respectively, resulting in the formation of angiogenic sprouts (Figure 2a). Cultures were stained with ActinRed (red), VE-cadherin (green), and Hoechst (blue). Sprout formation can be quantified over time by image analysis scripts in Image-J (Figure 2b). The sprouts are hollow as witnessed by confocal imaging of middle (i), top (ii), and cross-section (iii) (Figure 2c) and can be perfused with FITC-dextran dye (Figure 2d).
Prolonged exposure to the angiogenic cocktail causes angiogenic sprouts to anastomose, with a connection being formed between the two perfusion channels. Upon anastomosis, some angiogenic sprouts display the characteristic steps involved in pruning, while other angiogenic sprouts remained and increased their lumen diameter (figure 3).