Morphogen Gradients in Microfluidic Cultures

描述(由申请人提供)：该项目的长期目标是了解在发育过程中，形态原梯度如何形成大脑皮层和其他组织的模式。形态原梯度是动物发育的基础，而形态原缺陷是人类出生缺陷和皮质畸形的主要原因。尽管如此，关于形态梯度的作用机制仍然存在巨大的争议，这限制了我们对这些人类疾病的理解。在很大程度上，这场争论围绕着一个单一的问题--无法区分不依赖于细胞-细胞交流的形态生成活动(“经典”模型)和那些依赖于细胞-细胞交流的形态生成活动。到目前为止，对这个问题的洞察依赖于使用传统分离细胞培养的英勇研究，这些培养在实验效率和作为自然形态梯度的模型方面都是有限的。然而，微流控培养装置有可能解决这些限制。这种微型设备产生精确和连续的具有不同轮廓的生物分子梯度到细胞上，并被设计用于时间推移显微镜。这些特征应该会为形态梯度的建模和研究提供几个与传统培养相比的生物学和实用优势。对皮质前体细胞(CPC)的初步研究证实了该系统的前景，但也确定了需要优化的设备设计功能，以回答推动这一建议的基本问题-正常皮质中的哪些CPC反应完全由细胞外形态原浓度决定，哪些不是？ R21方案的目标是制造用于培养CPC的优化微流控设备(目标1)，并开发单细胞分辨率的实时分析方法，以便有效地研究CPC响应随形态原浓度、梯度分布、细胞密度和时间的函数(目标2)。为了实现这些目标，已经组建了一支在生物工程(原始梯度生成设备的开发商Noo Li Jeon)、形态生成梯度(Arthur Lander)和皮质开发(Edwin Monuki)方面拥有主要专业知识的多学科团队。如果成功，这项建议不仅应该促进我们对皮质发育和畸形以及一般形态原梯度的理解，而且还应该提供一种具有广泛基础和临床应用的多功能微流控工具。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how morphogen gradients pattern the cerebral cortex and other tissues during development. Morphogen gradients are fundamental to animal development, and morphogen defects are primary causes of human birth defects and malformations of the cortex. Nonetheless, tremendous controversy remains about the mechanisms by which morphogen gradients act, which limits our understanding of these human disorders. For the most part, this controversy revolves around a single issue - the inability to distinguish morphogen activities that do not depend on cell-cell communication (the "classical" model) from those that do. To date, insight into this issue has relied on heroic studies using traditional dissociated cell cultures, which are limited both in terms of experimental efficiency and as models of natural morphogen gradients. However, a microfluidic culture device has the potential to address these limitations. This microscale device generates precise and continuous biomolecular gradients with different profiles onto cells, and is designed for time-lapse microscopy. These features should provide several biological and practical advantages over traditional cultures for modeling and studying morphogen gradients. Preliminary studies with cortical precursor cells (CPCs) confirm the promise of this system, but have also identified device design features that need to be optimized in order to answer the basic question driving this proposal - which CPC responses in the normal cortex are determined solely by extracellular morphogen concentration, and which are not? The goals of this R21 proposal are to fabricate optimized microfluidic devices for culturing CPCs (Aim 1) and to develop real time assays with single cell resolution in order to efficiently study CPC responses as functions of morphogen concentration, gradient profile, cell density, and time (Aim 2). To achieve these goals, a multidisciplinary team with primary expertise in bioengineering (Noo Li Jeon, the developer of the original gradient-generating device), morphogen gradients (Arthur Lander) and cortical development (Edwin Monuki) has been assembled. If successful, this proposal should not only advance our understanding of cortical development and malformations, and of morphogen gradients in general, but should also provide a versatile microfluidic tool with a wide range of basic and clinical applications.