Subcellular Targeting of Dosant Streams in Culture

描述（由申请人提供）：二维（2D）培养物为在相对简单和可控的环境中研究神经元和其他细胞的特性提供了重要的模型系统。这种还原论方法的价值关键是在亚细胞，单细胞和细胞系综水平上扰动和表征神经元特性的技术的发展。虽然存在有价值的方法与细胞电子接口，目前的技术与神经元化学相互作用提供不足的空间控制，特别是在应用中，需要持续几秒钟或更长的时间段的剂量。该项目的总体目标是开发一种通用的方法，用于在多个站点并行地与神经元培养物进行化学接口，并对相互作用坐标进行任意实时控制。开发的方法将适用于神经科学及其他领域的广泛细胞生物学问题，包括趋化性和干细胞分化。具体来说，该项目的重点是开发一种方法，用于在膜内快速，激光介导引入孔，作为细胞生长的支持，以及分离两个流动室：细胞培养基室和底层dosant室。由薄膜屏障的点烧蚀引起的孔产生将来自高压剂量室的层流流引入细胞环境中。具体目标1是针对开发一个可重现的和良好表征的平台，重点是膜材料的评价和系统时空限制的表征。目标2的重点是通过实施孔的可逆阀门和修改流动-流方向性的手段来扩展平台的功能。目的3是针对该工具用于在多个感兴趣点并行刺激和调节细胞系和神经元培养物的效用的初步证明。

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.