Low Cost High Throughput Cell Cultivation Apparatus

描述（由申请人提供）：已经对几个物种进行了基因组测序。现在的重点是通过检查产生的蛋白质来彻底研究这些序列的编码。解释由数千个基因表达的数百万种蛋白质将需要组合的努力。细胞必须在受控制的环境中培养，并暴露于各种环境和生理条件下，才能引起不同的反应，从而阐明负责基因表达的因素。一个主要的限制是在受控条件下大量培养细胞的能力，这样就可以进行数千次实验。目前最先进的技术要求使用仪器化的生物反应器，这些反应器体积庞大，价格昂贵，不适合大量并行运行。虽然微量滴定板确实提供了大量实验的能力，但它们缺乏传感器和仪器来实现环境控制。

DESCRIPTION (provided by applicant): Several species have had their genomes sequenced. The focus is now shifting to a thorough study of what these sequences code for by examining the resulting proteins. Accounting for millions of proteins expressed by thousands of genes is going to require a combinatorial effort. Cells will have to cultured in controlled environments and exposed to a variety of environmental and physiological conditions in order to elicit differential responses so that the factors responsible for gene expression can be elucidated. A major limitation is the ability to grow cells under controlled conditions in large numbers such that thousands of experiments may be performed. The current state-of-the-art requires the use of instrumented bioreactors that are bulky and expensive and not amenable to being run in large numbers in parallel. Whereas microtiter plates do offer the ability for large numbers of experiments, they lack the sensors and instrumentation to enable environmental control. We are proposing to bridge this gap by utilizing low-cost, non-invasive optical sensor technology in order to make controlled cell cultivation possible in a system analogous to microtiter plates. Our goal is to make the monitoring and control of pO2, pH, and Optical Density (to measure cell growth) facile. Our approach is to use miniature scaled down versions (called microbioreactors) of liter scale bioreactors. We employ optical sensing techniques where an analyte-sensitive patch is placed in each microbioreactor where cells are grown and monitored optically from outside. In addition, we will equip the instrumentation for monitoring Green Fluorescent Protein (GFP), as this is a widely used marker of gene expression. The proposed system will allow 24 milliliter scale bioreactor experiments to be simultaneously conducted under precisely controlled conditions at about the same cost as a single laboratory bioreactor. Specifically in the first year (R21 period), we will: 1. Construct a cell cultivation platform consisting of 24 microbioreactors to be used with an instrument platform and equipped with sensors to monitor pO2, pH, Optical Density and GFP. 2. Demonstrate its use for simultaneous monitoring of 24 E. coli fermentations for the four parameters. These will also serve as our milestones for continuation onto the next phase. Over the next three years (R33 period), we will 3. Implement strategies for control of pO2, pH and timed nutrient addition. 4. Validate the system with a study of oxidative stress responses in E. coli using GFP as the reporter gene and demonstrate feasibility for mammalian cell culture. The above technology has the potential to significantly impact practices ranging from basic studies of gene expression to bioprocess development as pertaining to the development of pharmaceuticals and vaccines.