Cell Characterization and Separation Using Microfluidics for Biomedical Applications

博士论文有两个目的：第一是了解和设计微流体系统来分离培养中的利什曼原虫。二是利用该技术识别与细胞/生命周期阶段相关的生物标志物。利什曼原虫是一种原生动物寄生虫，影响着全世界数百万人。利什曼病主要表现为三种形式；内脏，皮肤和粘膜，导致身体畸形，残疾，甚至死亡。尽管这些感染可能非常广泛和危险，但就其生命周期和传播方式而言，它仍然是一种相对缺乏研究的寄生虫。这部分是由于在混合细胞群体中研究生命周期的每个阶段很困难。利什曼原虫具有复杂的生命周期，既需要媒介，也需要哺乳动物宿主。它们在两个寄主体内表现出几个不同的形态阶段，使它们能够生存、复制和感染。原刚体存在于沙蝇载体中，在传播到哺乳动物宿主之前，它们会通过多种细胞形式分化，在哺乳动物宿主中感染巨噬细胞并分化为复制性无尾刚体。一些proproprogote阶段是复制的，而另一些阶段是细胞周期阻滞的。在沙蝇体内的生命周期中，寄生体的长度会发生变化，先生长，再缩短，然后分裂。此外，处于生命周期不同阶段的细胞具有不同的鞭毛长度。由于利什曼原虫的细胞复制不同步，因此培养利什曼原虫会产生混合种群，因此人们对原毛菌的细胞分裂周期知之甚少，特别是在分子水平上。此外，目前还缺乏纯化不同细胞周期阶段的最佳方法，也缺乏识别它们的生物标志物。近年来，微流体螺旋通道已被用于从混合培养物中分离活寄生虫。这些通道迫使流体以高速通过微观通道，根据它们的大小、可变形性和形状分离细胞，精度高达96%。改变螺旋通道输出通道的几何形状、流体速率和位置会改变细胞的分离方式。本论文旨在根据利什曼原虫的大小、形状和可变形性来理解和设计分离利什曼原虫的螺旋通道，从而使利什曼原虫在其生命/细胞周期的每个阶段的纯种群分离成为可能。有了纯种群，我们将能够更好地研究它们的分子差异，并确定一套独特的生物标志物，特定于每个生命/细胞周期阶段，这将有助于研究利什曼原虫的复制、生命周期和传播。本博士的工作将推动两个领域的研究。首先，它将为利什曼原虫细胞周期的基本生物学和分子控制提供信息，使其能够与其他着丝质体寄生虫进行比较。其次，它将使人们更深入地了解微流体在细胞分离和样品制备中的应用。这两个领域对长期利益都有更广泛的影响。例如，利什曼原虫细胞/生命周期生物标志物的鉴定将允许在未来的利什曼原虫研究中进行更精确的研究，而对微流体的更好理解不仅可以用于分离寄生虫，还可以用于分离细胞、细菌和其他感染因子。因此，这项技术在研究和最终诊断方面具有很大的潜力；然而，更好地理解它是如何工作的是必要的，以利用这些好处。拟议的研究将有助于理解这项技术。

The proposed PhD thesis has two aims: the first is to understand and design microfluidic systems to separate Leishmania parasites in culture. The second is to use this technology to identify biomarkers correlating to their stage in their cell/life cycle. Leishmania is a protozoan parasite that affects millions of people worldwide. Leishmaniasis manifests in three main forms; visceral, cutaneous and mucocutaneous, leading to physical deformities, disabilities, and even death. Despite how widespread and dangerous these infections can be, it is still a relatively understudied parasite with regards to its life cycle and mode of transmission. This is partly due to the difficulty in studying each stage of the life cycle in mixed populations of cells. Leishmania parasites have a complex life cycle requiring both vector and mammalian hosts. They exhibit several distinct morphological stages within the two hosts allowing them to survive, replicate and infect. Promastigotes are found in the sand fly vector and differentiate through multiple cell forms before they are transmissible to a mammalian host, where they infect macrophages and differentiate into replicative amastigotes. Some promastigote stages are replicative while others are cell cycle arrested. During the life cycle within the sand fly, the parasite length changes, growing and then shortening before division. Additionally, cells at different stages of the life cycle have varying flagellum length. Little is understood about the cell division cycle of promastigotes, particularly at the molecular level, because culturing Leishmania results in mixed populations due to their asynchronous cell replication. Furthermore, there is a lack of optimal methods to purify the different cell cycle stages and a paucity of biomarkers to identify them. Recently, microfluidic spiral channels have been used to separate live parasites from mixed cultures. These channels force fluid through microscopic channels at high velocities, which separate cells based on their size, deformability and shape, with accuracies of up to 96%. Altering the geometries, fluid rates and location of the output channels of the spiral channels alters how the cells are separated. This thesis would aim to understand and design spiral channels for the separation of Leishmania parasites based on their size, shape and deformability, allowing the isolation of pure populations of Leishmania at each stage of its life/cell cycle. With pure populations we would be able to better study their molecular differences and identify a unique set of biomarkers specific to each life/cell cycle stage, which would then facilitate studies on Leishmania replication, life cycle and transmission.The work carried out in this PhD would progress two areas of research. Firstly, it would inform on the basic biology and molecular control of the cell cycle of Leishmania, allowing for its comparison against other kinetoplastid parasites. Secondly, it would give a deeper understanding of the uses of microfluidics in cell separation and sample preparation. Both of these areas have wider implications for long term benefits. For example, the identification of Leishmania cell/life cycle biomarkers would allow for more precise research in future Leishmania studies, while a better understanding of microfluidics could be used to separate not just parasites, but cells, bacteria and other infectious agents. This technology therefore has much potential in research and eventually diagnostics; however, better understanding of how it works is necessary to harness these benefits. The proposed research would aid in the understanding of this technology.