【作者】 张洁；

【导师】 林金明；

【作者基本信息】 清华大学 ， 化学， 2016， 博士

【摘要】 微流控芯片技术因其具有的一系列优点,如样品试剂消耗少、结构功能多样化、集成化程度高、与细胞尺度接近等优点,近来广泛应用于细胞相关研究领域。通过结合不同的分析检测方法以及集成不同的功能结构单元,取得了显著的研究进展。其中相对传统方法,微流控芯片最大的优势是其集成化的功能,能够将多种不同的细胞或组织有序地整合为一个体系,而这也是当前细胞相关研究的一个重要发展方向,将体内状态下相关的多种细胞共培养并进行相互作用,可以更好地保持细胞的功能及生物学特性,这对于建立更完善的体外生物模型有至关重要的意义。在本论文中,我们围绕微流控芯片上细胞共培养开展了一系列研究。首先,我们对微流控技术的发展现状及在细胞研究中的应用进行了综述,其中着重介绍了在细胞共培养及生物微环境模拟中的的工作,分析论证了微流控芯片上进行细胞共培养的优势和意义。二、在微流控芯片上共培养了肝癌细胞和乳腺癌细胞,建立了一种微流控芯片上的肝-肿瘤模型,成功实现了前体药物卡培他滨的代谢与作用,并与质谱联用对原药及中间代谢产物进行了检测。使用这一平台,不仅可以对药物作用过程中的细胞状态进行活性检测、增殖检测及显微镜直接观察,同时可以使用质谱对系统中的信号分子或中间产物进行检测与鉴定。三、发展了一种使用inkjet在微流控芯片内对不同细胞进行微量精准定位共培养的方法,成功地将肝癌细胞和胶质瘤细胞同时打印并整合至微流控芯片内,用于前体药物的代谢及扩散实验。这一方法可以大幅提高微流控芯片内多种细胞精确定位共培养的效率,具有方便高效、易于自动化等优点。四、通过在微流控芯片中共培养胶质瘤细胞、内皮细胞及巨噬细胞,对胶质瘤微环境进行了一定程度的模拟,并对微环境中细胞间相互作用及细胞形态变化、表型变化、迁移情况进行同时监测,观察到了肿瘤细胞在微环境中与体内状况相一致的上皮-间充质转化的现象,同时也检测到了细胞的迁移运动、及通过细胞因子变化对微环境中巨噬细胞表型变化进行分析。更多还原

【Abstract】 Recently microfluidic chips have been widely used in cell research,for a lot of advantages over conventional methods like low sample and reagent consumption,versatile structures and functions,high integration,similar size to cells and in vivo microenvironment.Tremendous advances have been achieved through combining microfluidic technology with different analysis methods and integrating various structures and functions.Compared to conventional methods,the primary advantage of microfluidic chips is high integration,enabling the combination of different cells or tissues into an ordered system,which is also an important trend in current cell related research.By co-culturing different cells that are related for in vivo situation,the functions and biological characters could be better retained,which is of great significance for the reconstruction of in vitro biological models.In this thesis,some works around cell co-culture and biological environment imitation were carried out.First,the development of microfluidic technology and the applications in cell research were summarized.Specially works about cell co-culture and biological environment imitation were emphatically introduced and discussed,showing the advantages and significance of cell co-culture research on microfluidic chips.Second,an in vitro liver-tumor model was established in the microfluidic chip by co-culturing liver cancer cells and breast cancer cells.The prodrug capecitabine metabolism was successfully realized and the intermediate metabolite was successfully detected by mass spectrometry.Using this platform not only the cell vitality and proliferation could be real time monitored,the signal molecules or intermediate product could also be detected and identified by MS,showing a potential to replace conventional drug screening methods.Third,a novel approach for precisely controlled microscale cell patterning and co-culture in microfluidic chip by inkjet printing was successfully developed.Liver cancer cells and glioma cells were successfully co-patterned in the microchip and realized drug metabolism and diffusion experiment.The developed approach will help signi?cantly increase cell patterning efficacy in microfluidic chip as well as reduce theextent of laborious experimental work by automation.Fourth,a multi-functional 3D microfluidic platform was developed for co-culture of glioma cells,endothelial cells and macrophages to imitate the glioma niche at some extent.The cell morphology change,phenotype change and migration in the microenvironment could be simultaneously monitored.EMT change of glioma cells were observed under co-culture,which was consistent with in vivo situation.Cell migration and phenotype change of macrophages indicated by cytokines secretion change were also observed.更多还原