三维液滴链阵列用于细胞迁移和多组织共培养研究

发布时间：2018-04-07 19:04

本文选题：液滴链　切入点：液滴微流控　出处：《浙江大学》2016年博士论文

【摘要】：构建体外细胞迁移和共培养模型将有利于药物筛选、早期疾病诊断和基础生物学的研究。微流控系统在芯片结构的设计、细胞培养的材料、流体控制的方法上都具备多种选择,可以用于细胞微环境的控制和模拟,实现细胞三维培养和共培养,进而用于研究细胞行为、探究病理生理现象、开发和筛选新的药物。然而大多用于细胞迁移行为和共培养研究的微流控装置受芯片复杂性和流体控制装置的限制,难以适用于多种细胞生物学研究。本工作的目的是建立一个简单的多模式液滴微流控方法,利用基于多孔膜的液滴链装置完成多种细胞实验,用于低消耗、高通量的细胞生物学研究,包括多模式细胞迁移的研究和多细胞共培养模型的建立。第一章我们综述了应用于细胞迁移、细胞三维培养、细胞共培养的微流控芯片技术的研究进展。物质浓度梯度的形成对于细胞迁移的研究至关重要,我们主要分类介绍了基于多相层流、微通道、凝胶、多孔膜体系等构建的浓度梯度生成系统及其在细胞迁移实验中的应用。在本章中,我们还介绍了微流控三维细胞培养的主要方法,包括利用悬滴和凝胶等方法实现细胞的三维生长；细胞共培养的方法则主要介绍了图案化的细胞共培养方法和基于膜的与基于通道的细胞共培养方法；以及利用细胞三维培养、共培养技术构建生理相关的多器官组织模型的进展。在第二章中,我们建立了一种基于多孔膜的三维液滴链阵列系统用于多模式细胞迁移的研究。三维液滴链通过多孔膜上的微孔进行物质交流,通过PDMS通孔阵列片控制膜上下液滴的相对位置,改变液滴的体积、位置、内容物可以控制物质扩散速度,生成不同的浓度梯度,进行多种模式的细胞迁移实验。液滴链中液滴的体积在500-800 nL之间,一个芯片上可完成高达81个细胞迁移实验。我们将这一细胞迁移系统应用于多种模式的细胞迁移实验中,包括精准的细胞迁移实验、竞争性细胞迁移实验、仿生趋化性细胞迁移实验。在第三章中,基于第二章的方法,我们设计了集成度更高的花型液滴链和珠串型液滴链芯片。在花型液滴链中,将悬滴三维培养、多细胞共培养的方法用于多种细胞相互作用的研究中,构建了集成肝、肺、肾、肠、心等细胞的多器官液滴链阵列芯片,并应用于转移性乳腺癌细胞MDA-MB-231向不同器官微组织的迁移倾向性研究。初步构建了基于生理条件建立的珠串型液滴链芯片,以模拟药物在体内的代谢过程。
[Abstract]:The establishment of cell migration and co-culture model in vitro will be beneficial to drug screening, early disease diagnosis and basic biology research.The microfluidic system has many choices in chip structure design, cell culture materials and fluid control methods. It can be used to control and simulate cell microenvironment and realize cell three-dimensional culture and co-culture.It can be used to study cell behavior, explore pathophysiological phenomena, and develop and screen new drugs.However, most of the microfluidic devices used in cell migration and co-culture are limited by chip complexity and fluid control devices, which are difficult to be applied to many cell biology studies.The purpose of this work is to establish a simple multi-mode droplet microfluidic method, and to use the liquid droplet chain device based on porous membrane to complete a variety of cell experiments, which can be used in low consumption and high throughput cell biology research.Including the study of multi-model cell migration and the establishment of multi-cell co-culture model.In the first chapter, we review the research progress of microfluidic chip technology used in cell migration, cell three-dimensional culture and cell co-culture.The formation of concentration gradient is very important to the study of cell migration. We mainly introduce the concentration gradient generation system based on multiphase laminar flow, microchannel, gel and porous membrane system and its application in cell migration experiments.In this chapter, we also introduce the main methods of microfluidic three-dimensional cell culture, including the use of suspension and gel methods to achieve three-dimensional cell growth;The methods of cell coculture mainly include patterned cell coculture, membrane-based and channel-based co-culture, and three-dimensional cell culture.Progress of co-culture techniques in the construction of physiological-related multi-organ tissue models.In the second chapter, we establish a three-dimensional droplet chain array system based on porous membrane for the study of multi-mode cell migration.The three-dimensional droplet chain communicates through the micropores in the porous membrane, and the relative position of the droplets up and down the membrane is controlled by the PDMS through pore array. The volume, position and contents of the droplets can control the diffusion velocity of the material and produce different concentration gradients.Cell migration experiments were carried out in a variety of models.The volume of droplets in the droplet chain ranges from 500 to 800 NL, and up to 81 cell migration experiments can be performed on a single chip.We applied this cell migration system to many cell migration experiments, including accurate cell migration, competitive cell migration, and bionic chemotactic cell migration.In Chapter 3, based on the method in Chapter 2, we design a more integrated droplet chain and a droplet chain chip.In the floral droplet chain, the method of suspension three-dimensional culture and multicellular co-culture was used in the study of multi-cell interaction, and a multi-organ droplet chain array of liver, lung, kidney, intestine, heart and so on was constructed.It was used to study the migration tendency of metastatic breast cancer cell line MDA-MB-231 to different organ microtissues.A series of droplet chain chips based on physiological conditions were constructed to simulate the drug metabolism in vivo.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q813

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