This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.