A Cellular Chip for High-Throughput Measurements of Single, Growing Cells

用于单个生长细胞高通量测量的细胞芯片

• 批准号: 8374248
• 负责人: Philippe Cluzel
• 金额: $ 29.85万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8374248
• 关键词: Antibiotics; Bacteria; Biological; Buffers; Cell Count; Cell Density; Cell Fraction; Cells; Complex; Computer software; Crowding; DNA Microarray Chip; Data; Data Set; Development; Devices; Diffusion; Environment; Flagella; Fluorescence; Gel; Gene Expression; Generations; Genome; Growth; Health; Heterogeneity; Human; Hydrogels; Image; Image Analysis; Individual; Intervention; Laboratories; Life; Measurement; Measures; Mechanics; Methods; Microarray Analysis; Microbiology; Microfluidics; Microscope; Microscopy; Monitor; Nutrient; Pattern; Printing; Property; Proteins; Recording of previous events; Reporter; Resolution; Running; Sepharose; Solid; Speed; Starvation; Stress; Structure; Techniques; Testing; Time; Work; analog; base; cellular imaging; charge coupled device camera; density; design; genome wide association study; imaging modality; improved; lithography; method development; movie; nanopattern; novel; porous hydrogel; pressure; research study; response; statistics; submicron; tool

DESCRIPTION (provided by applicant): In this proposal, we will develop a platform for the high-throughput measurement of growth and gene expression in single bacterial cells. Time-lapse microscopy of growing bacteria has been an extremely successful technique, revealing the natural heterogeneity that underlies growth and gene expression in single cells. However, the exponential growth of bacteria quickly overwhelms the solid support, depleting the local nutrient environment, crowding cells, and limiting measurement duration. Here, we will circumvent the low throughput of such measurements by combining microfluidic techniques, microarray technology, and automated image analysis to create a massively parallel, high-throughput, single-cell 'chip'-a living analog of the DNA microarray. In our first aim, we will continue our construction of a single-cell chemostat-a micro-patterned pad that can be used to cultivate a high density of cells for long durations while allowing individual cells to be imaged. We use soft-lithography to create nano-patterned hydrogel pads that constrain bacteria to high-density linear tracks. Buffer flow through microfluidic lines in the gel delivers fresh nutrient bufer and washes away excess cells. Soft and porous hydrogels hold cells in place with a non-perturbative pressure and allow unrestricted diffusion to maintain a uniform nutrient environment. We will focus on creating a device that allows real-time control over the nutrient environment and precise control over the mechanical properties of the hydrogel that holds the cells. This single-cell chemostat will dramatically increase both the duration of time-lapse measurements and the number of cells that can be imaged in a field-of-view. In our second aim, we will develop methods to leverage existing microarray technology to print thousands of distinct bacterial strains onto a single patterned hydrogel. We will pursue two approaches for printing cells to patterned pads, both of which have been validated by proof-of-principle experiments. Advances in this aim will allow multiple experiments to be run in parallel on a single patterned hydrogel, providing a dramatic increase in the number of distinct bacterial strains that can be characterized simultaneously. In our final aim, we will develop the necessary image-based techniques to perform high-speed, high- throughput, time-lapse microscopy of growth on a nano-patterned pad, and we will develop the automated image analysis software needed to extract the full history of growth, division, and fluorescent gene expression within cellular lineages from these large data sets. These techniques will allow us to monitor the entire life of millions of cells in a single overnight measurement. These advances will make high-throughput, single-cell studies possible in our laboratory. We will be able to characterize the transcriptional network that regulates the assembly of complex, multi-protein machines such as the flagella; and investigate the transcriptional response to multiple antibiotic stresses-a proble that is extremely relevant to human health. Moreover, by providing a platform for high-throughput single-cell measurements, our cellular chip will prove a useful tool for answering many pressing questions in microbiology. PUBLIC HEALTH RELEVANCE: We will develop a single-cell chemostat that allows the high-throughput measurement of growth and gene expression of single living cells growing under constant nutrient conditions. We will develop methods to print thousands of distinct strains to a single device and methods to collect and analyze massive time-lapse data sets of bacteria growing in this device. This experimental platform will allow us to study genome-wide transcriptional response to a wide range of environmental stresses, including multiple antibiotic exposure and nutrient starvation, at the single-cell level.

描述（由申请人提供）：在此提案中，我们将开发一个平台，用于对单个细菌细胞中生长和基因表达进行高通量测量。生长细菌的延时显微镜是一种非常成功的技术，揭示了单个细胞中生长和基因表达的自然异质性。但是，细菌的指数生长迅速淹没了固体支持，耗尽了局部营养环境，拥挤的细胞和限制测量持续时间。在这里，我们将通过结合微流体技术，微阵列技术和自动图像分析来规避此类测量值的低通量，从而创建了一个大量平行的，高通量的，单细胞的单细胞'chip'-a-a Living live live abalog。 在我们的第一个目标中，我们将继续构建单细胞化学固定的微图案垫，该垫可用于长时间培养高密度的细胞，同时允许单个细胞成像。我们使用软线学来创建纳米图案的水凝胶垫，从而将细菌限制为高密度线性轨道。缓冲液通过凝胶中的微流体线流动可提供新鲜的营养粉末，并洗掉多余的细胞。软和多孔水凝胶将细胞固定在适当的位置，并允许不受限制的扩散以维持均匀的营养环境。我们将专注于创建一种设备，该设备允许对养分环境进行实时控制，并精确控制容纳细胞的水凝胶的机械性能。这种单细胞化学仪表板将大大增加延时测量的持续时间和可以在视野中成像的细胞数量。 在我们的第二个目标中，我们将开发方法来利用现有的微阵列技术将数千种不同的细菌菌株打印到单个图案水凝胶上。我们将采用两种方法来打印细胞对图案垫，这两种方法均已通过原理实验验证。此目标的进步将允许多个实验在单个图案水凝胶上并联运行，从而使可以同时表征的不同细菌菌株的数量显着增加。 在我们的最终目标中，我们将开发必要的基于图像的技术，以在纳米体模式垫上执行高速，高吞吐量，延时显微镜的生长显微镜，我们将开发从这些大型数据集中从细胞谱系中提取完整的生长，分裂和荧光基因表达中荧光基因表达的完整历史所需的自动图像分析软件。这些技术将使我们能够在一次过夜测量中监视数百万个细胞的整个寿命。 这些进步将使我们的实验室中的高通量，单细胞研究成为可能。我们将能够表征调节复杂的多蛋白机（例如鞭毛）组装的转录网络；并研究对多种抗生素应激的转录反应 - 与人类健康非常相关的特征。此外，通过为高通量单细胞测量提供一个平台，我们的蜂窝芯片将证明是回答微生物学中许多紧迫问题的有用工具。 公共卫生相关性：我们将开发一个单细胞化学仪，该化学仪，允许对恒定营养条件下生长的单个活细胞的生长和基因表达进行高通量测量。我们将开发将数千种不同菌株打印到单个设备的方法和方法，以收集和分析该设备中生长的细菌的大量延时数据集。这个实验平台将使我们能够在单细胞水平上研究各种环境应力的全基因组转录反应，包括多种抗生素暴露和营养饥饿。