Abstract 17423: A Novel in vitro Platform of Perfused Human Microtissues for the Study of Neovascularization

Abstract

Understanding the process of vascular network formation and remodeling is key to designing effective strategies for the treatment of ischemic vascular diseases. Much of our current understanding of the neovascularization process is limited to in vivo studies, which require extrapolation of results to human, or in vitro models that lack the dynamic feature of perfusion. In this work we report the development of an in vitro model system of metabolically active microtissues that exchange nutrients and waste through perfused capillaries. For such design, a device consisting of 2 fluidic channels on either side of a central tissue channel, containing fibroblasts seeded in a fibrin matrix (Fig 1A), was microfabricated in polydimethylsiloxane. To study vasculogenic-like processes, ECs were co-seeded with the fibroblasts in the tissue channel. The devices were grown at 5% oxygen tension and formation of perfused vessel networks was encouraged by mechanical (pressure gradients, interstitial flow) and chemical stimuli (hypoxia and nutrient deprivation). Vessel network formation was observed within a week of culture and as early as day 1. Perfusion of the network was confirmed at day 18. Fluorescent dextran (70kDa) introduced on one end of one of the fluidic channel (Fig 1B-C) was shown to reach the other side of the device by traveling through the capillaries of the microtissues as evidence by the presence of dextran in the tissue channel and the absence of dextran in the fluidic channel. (Fig 1D) Anastomosis of vessels in the tissue channel with the fluidic channels was verified by tracking movement of fluorescent 1µm beads introduced into the fluidic channel and through patent capillaries (20-105 µm/s) in the tissue channel. Confocal microscopy of devices stained for CD31 (EC marker) and DAPI (nuclear marker) showed the extensive vessel network (Fig 1E) and further demonstrated the presence of capillary lumens containing microbeads (Fig 1F).