Anti-angiogenic therapy, which suppresses tumor growth by disrupting oxygen and nutrient supply from blood to the tumor, is now widely accepted as a treatment for cancer. To investigate the mechanisms of action of these anti-angiogenesis drugs, new three dimensional (3D) cell culture-based drug screening models are increasingly employed. However, there is no in vitro high-throughput screening (HTS) angiogenesis assay that can provide uniform culture conditions for quantitative assessment of physiological responses to chemoattractant reagents under various concentrations of anti-angiogenesis drugs. Here we describe a method for screening and quantifying the vascular endothelial growth factor (VEGF)-induced chemotactic response on human umbilical vein endothelial cells (HUVECs) cultured under different concentrations of bortezomib, a selective 26S proteasome inhibitor. With this quantitative microfluidic angiogenesis screen (QMAS), we demonstrate that bortezomib-induced endothelial cell death was preceded by a series of morphological changes that develop over several days. We also explore the mechanisms by which bortezomib can inhibit angiogenesis.

We developed a computational model to predict oxygen levels in microfluidic plastic devices during cell culture. This model is based on experimental evaluation of oxygen levels. Conditions are determined that provide adequate oxygen supply to two cell types, hepatocytes and endothelial cells, either by diffusion through the plastic device, or by supplying a low flow rate of medium.

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Hepatic stellate cells (HSCs) form a functional unit with endothelia and hepatocytes in the liver to play a pivotal role in heterotypic cellular communication. To investigate this role of HSCs, it is of great benefit to establish a triculture model that forms the functional unit from proximal layers of hepatocytes, HSCs, and endothelial cells (ECs). Here, we established a three-dimensional triculture model, using a microporous membrane to create the functional unit. HSC behavior was controlled by the membrane pore size, which was critical for achieving proximal cell layers. With a specific pore size, the HSCs intercalated between layers of hepatocytes and ECs, due to the limitation on HSC behavior. When only cytoplasmic processes of quiescent HSCs were adjacent to ECs, while the HSC bodies remained on the side of the hepatocytes, the ECs changed morphologically and were capable of long-term survival. We confirmed that HSCs mediated the communication between hepatocytes and ECs in terms of EC morphogenesis. This triculture model allows us to investigate the roles of HSCs as both facilitators and integrators of cell-cell communication between hepatocytes and ECs, and is useful for investigating heterotypic cellular communication in vitro.