Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

This article reports a microfluidic device for the generation of stable, steady-state, user-defined concentration profiles for the long-term maintenance of stable diffusion gradients. The microinstrument allows both dynamic temporal and dynamic spatial control over userdefined concentrations and concentration gradients of multiple chemicals. With this device, one can create an in vitro environment capable of approximating the complex in vivo biological condition for cellular studies. In addition, the device has potential application in combinatory drug discovery, electrophoretic applications, ligand binding, etc. 3D computer simulations and analysis of arbitrary concentration profiles are presented along with experimental validation using multiple diffusing species.

Embryonic development of the spinal cord proceeds through a carefully orchestrated temporal and spatial sequence of chemical cues to provide precise patterning of adult cell types. Recreating this complex microenvironment in a standard cell culture dish is difficult, if not impossible. In this paper, a microfluidic device is used to recapitulate, in vitro, the graded patterning events which occur during early spinal cord development. The microdevice design is developed using COMSOL modeling, with which the spatiotemporal profiles of multiple, diffusible morphogens are simulated. Four independently addressed source/sinks are employed to generate two overlapping orthogonal gradients within a cell culture chamber, mimicking the dorsoventral and anteroposterior axes of the developing embryo. Mouse embryonic stem cells are directed therein to differentiate into motor neurons in a spatially organized manner, reminiscent of a neural tube.