Collaborative Research: Integrated Microfludic Platform for High Throughput Single Cell Gene Profiling

人类细胞中的基因表达是将基因中嵌入的信息转化为细胞产品的合成的过程。它是细胞和组织正常和病理发育的关键方面。目前的批量基因表达分析依赖于从多个细胞或组织样本中提取的分子，因此包含不同程度的细胞异质性。因此，很难通过这些“细胞群体平均”测量来确定不同发育阶段的基因之间的调控关系。研究人员此前已经证明，微流控技术可以从单细胞中提取总信使核糖核酸(MRNA)，并在同一芯片上合成互补脱氧核糖核酸(CDNA)，从而高效地进行单细胞基因表达谱分析，并减少可检测到的最小信使核糖核酸分子数量。然而，微流控器件目前面临的挑战之一是难以使用气动泵阀机构进行单细胞寻址，这是一项耗时和劳动密集型的任务。在这个项目中，将开发一个用于基因图谱应用的大规模并行单细胞mRNA分析的微流控平台。该装置在一个聚二甲基硅氧烷(PDMS)微流控芯片上集成了三个功能区域：一个高速微型荧光激活细胞分选器(µFACS)，用于大规模并行单细胞操作的光电镊子(OET)，以及用于单细胞mRNA提取和基因转化的1000个微流控孔。该设备将解决将OET与微流控设备集成以实现大规模并行单细胞操作的技术问题。1000个细胞将被单独捕获并输送到微流控井中，在那里细胞被分裂或裂解，用于基因图谱分析。集成OET可以消除在单细胞操作中被证明效率极低的多路复用微流控网络，取而代之的是可实时重新配置的动态光学图像。这一提议的成功将实现一种低成本、完全集成的微流控芯片，能够在1000个单细胞上进行大规模并行基因图谱分析。在这个项目的过程中发展起来的知识将被纳入调查人员？本科生和研究生的教学活动。这项提案的结果将在国际会议和同行评议的期刊上发表，调查员网站上也将提供信息。少数民族研究生和本科生将通过自主研究课程参与这些项目。参与该项目的学生将接触到南加州大学医学院和加州大学洛杉矶分校工程学院之间良好的多学科培训环境。PI还将参与加州大学洛杉矶分校(UCLA)的杰出外联计划(CEED)，招募人数不足的大学生，以建立一个“虚拟化学实验室”，让世界上每个人都能通过互联网控制研究实验室中的细胞。

Gene expression in human cells is the process that translates the information embedded in agene into the synthesis of a cellular product. It is a critical aspect of both normal and pathological development of cells and tissues. Current bulk gene expression assays rely on molecules extracted from multiple cells or tissue samples, therefore containing various degrees of cellular heterogeneity. As a result, it is difficult to determine the regulatory relationship of genes in different phases of development from these 'cell population averaging' measurements. The investigators have previously demonstrated that a microfluidic technology can be used for extracting total messenger Ribonucleic acid (mRNA) from single-cells and synthesizing complementary Deoxyribonucleic acid (cDNA) on the same chip for high efficiency single-cell gene expression profiling and reduces the minimum detectable number of mRNA molecules. However, one of the current challenges of them microfluidic device is the difficulty of using pneumatic pumping and valving mechanism for single cell addressing, which is a time-consuming and labor intensive task. In this project a microfluidic platform will be developed for massively parallel single cell mRNA analysis for gene profiling applications. The proposed device integrates three functional regions on a single Polydimethylsiloxane (PDMS) microfluidic chip: a high speed microscale fluorescence activated cell sorter (µFACS), optoelectronic tweezers (OET) for massively parallel single cell manipulation, and 1000 microfluidic wells for single cell mRNA extractions and cDNA conversion. This device will solve the technical issues in integrating OET with microfluidic devices to enable massively parallel single cell manipulation. One thousand cells will be individually trapped and transported into microfluidic wells where cells are split, or lysed, for gene profiling analysis. Integrating OET allows eliminating the multiplexed microfluidic control network that has been proven extremely inefficient in single cell manipulation and replacing it with dynamic optical images that can be reconfigured in real-time. Success in this proposal will realize a low-cost, fully integrated microfluidic chip capable of conducting massively parallel gene profiling on 1000 single cells. Knowledge developed during the course of this project will be incorporated into the investigators? teaching activities at both the undergraduate and graduate levels. Results of this proposal will be published in international conferences and peer-reviewed journals and information will also be available on the investigator website. Minority graduate and undergraduate students will participate in these projects through independent research courses. Students involved in this project will be exposed to an excellent multidisciplinary training environment between the USC Medical School and UCLA Engineering School. The PI will also be involved with the outstanding outreach program (CEED) in UCLA to recruit underrepresented college students for constructing a 'Virtual Chemical Lab' allowing everyone in the world to control cells in investigators' lab through Internet.