Subcellular Targeting of Dosant Streams in Culture

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DESCRIPTION (provided by applicant): Two-dimensional (2D) cultures provide important model systems for studying properties of neurons and other cells in relatively simple and controllable environments. Key to value of this reductionist approach is development of technologies for perturbing and characterizing neuronal properties at the subcellular, single-cell, and cellular-ensemble levels. Although valuable methods exist for interfacing with cells electronically, current techniques for interacting chemically with neurons provide inadequate spatial control, particularly in applications that require dosings to be sustained for periods of seconds or longer. The overall objective of this project is to develop a generalized approach for chemically interfacing with neuronal cultures at numerous sites in parallel and with arbitrary, real-time control over interaction coordinates. The methods developed will be applicable to a broad range of cell biology problems in neuroscience and beyond, including chemotaxis and stem-cell differentiation. Specifically, the project is focused on development of an approach for rapid, laser-mediated introduction of apertures within membranes that serve as supports for cell growth as well as separating two flow chambers: a cell-medium chamber and an underlying dosant chamber. Aperture creation, caused by point ablation of the thin membrane barrier, introduces a laminar-flow stream from the higher pressure dosant chamber into the cell environment. Specific Aim 1 is directed toward the development of a reproducible and well-characterized platform, with a focus on evaluation of membrane materials and characterization of spatiotemporal limits of the system. Aim 2 focuses on expanding the functionality of the platform through the implementation of reversible valving of apertures and a means to modify flow-stream directionality. Aim 3 is directed toward initial demonstration of the utility of this tool for stimulating and modulating cell-line and neuronal cultures at multiple points-of-interest in parallel.