Microfluidic-based 3D cell culture for studies of biophysical and biochemical regulation of endothelial function

Abstract

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigations. The ultimate challenge lies in replicating the native cell/tissue environment ex vivo. Certain key features of living tissues such as the three dimensionality, biophysical and biochemical microenvironment cannot be readily replicated in traditional culture platforms. Moreover, the capability for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small populations is lacking. In this thesis, we developed a novel multiparameter microfluidic-based cell culture platform. The system permits 2D or 3D culture of cells on/in biologically-derived or synthetic hydrogel scaffolds and allows for controlled flow rates, pressure and concentration gradients while directly visualizing cellular response. In addition to the realtime and post-fixation imaging using optical microscopy, methods were developed to extend post-fixation analysis to transmission electron microscopy (TEM). The platform was subsequently used to demonstrate for the first time, two microfluidicbased 3D in vitro assays with direct relevance to tumor development and glaucoma. For the first assay, biochemical induced sprouting was demonstrated. Endothelial cells sprout from an intact monolayer to form multicellular capillary-like structures. Furthermore, using time-lapse microscopy the cellular dynamics during sprouting angiogenesis were observed with great detail, showing tip cell dynamics, cell division events and lumen formation. Of particular relevance to tissue engineering community, we demonstrated that endothelial cells when cultured for several days can assemble into vascular networks with open, perfusable lumen. Using this new system, we present novel findings and results supporting a potential mechanism for flow-mediated mechanical regulation of angiogenesis by transendothelial fluid flow. We demonstrate that flow direction is sufficient to define an angiogenic ON or OFF state. The balance is tipped by forces generated at mechano-sensitive cell-matrix adhesions involving FAK-mediated signaling. These results provide one explanation for the bias towards angiogenesis occurring from the venous side of the circulation. For the second assay, an aqueous humor (AH) outflow model was developed. Subsequent proof-of-concept experiments confirmed its capability for studying the role of the inner wall endothelium in the regulation of AH outflow dynamics.