Subcellular Targeting of Dosant Streams in Culture

描述（由申请人提供）：二维（2D）培养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。这种还原论方法的关键价值在于在亚细胞、单细胞和细胞集合水平上对神经元特性进行扰动和表征的技术发展。尽管存在与细胞电子连接的有价值的方法，但目前与神经元化学相互作用的技术无法提供足够的空间控制，特别是在需要持续数秒或更长时间的剂量的应用中。该项目的总体目标是开发一种通用的方法，在并行和任意实时控制相互作用坐标的情况下，在许多地点与神经元培养物进行化学接口。所开发的方法将适用于神经科学和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快速、激光介导的膜内孔引入方法，作为细胞生长的支持，以及分离两个流动室：细胞-介质室和底层剂量室。由薄膜屏障的点烧蚀引起的孔径产生，将高压剂量室的层流引入细胞环境。具体目标1是针对一个可重复和表征良好的平台的发展，重点是膜材料的评估和表征系统的时空限制。目标2侧重于通过实现孔径可逆阀和改变流体方向的方法来扩展平台的功能。目标3旨在初步演示该工具在多个兴趣点平行刺激和调节细胞系和神经元培养的实用性。

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.