Cell Characterization and Separation Using Microfluidics for Biomedical Applications

拟议的博士论文有两个目标：第一个是理解和设计微流体系统，以分离培养中的利什曼原虫寄生虫。第二个是使用这项技术来识别与其细胞/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，影响全世界数百万人。利什曼病表现为三种主要形式;内脏，皮肤和粘膜皮肤，导致身体畸形，残疾，甚至死亡。尽管这些感染是多么广泛和危险，但就其生命周期和传播方式而言，它仍然是一种相对欠研究的寄生虫。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的困难。利什曼原虫具有复杂的生命周期，需要载体和哺乳动物宿主。它们在两种宿主中表现出几个不同的形态学阶段，使它们能够存活，复制和感染。前鞭毛体存在于白蛉载体中，并在它们可传播至哺乳动物宿主之前通过多种细胞形式分化，在哺乳动物宿主中它们感染巨噬细胞并分化成复制型无鞭毛体。一些前鞭毛体阶段是复制的，而另一些则是细胞周期停滞的。在白蛉的生命周期中，寄生虫的长度会发生变化，在分裂之前会变长，然后变短。此外，处于生命周期不同阶段的细胞具有不同的鞭毛长度。关于前鞭毛体的细胞分裂周期，特别是在分子水平上，人们了解得很少，因为培养利什曼原虫会导致混合种群，这是由于它们的异步细胞复制。此外，缺乏纯化不同细胞周期阶段的最佳方法，也缺乏鉴定它们的生物标志物。最近，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，根据细胞的大小，变形能力和形状分离细胞，准确率高达96%。改变螺旋通道的输出通道的几何形状、流体速率和位置改变了细胞如何被分离。本论文的目的是了解和设计螺旋通道分离利什曼原虫寄生虫的大小，形状和变形能力的基础上，允许在其生命/细胞周期的每个阶段分离的纯群体的利什曼原虫。与纯人群，我们将能够更好地研究他们的分子差异，并确定一套独特的生物标志物，具体到每个生命/细胞周期阶段，这将促进利什曼原虫复制，生命周期和传播的研究。在这个博士进行的工作将进展两个领域的研究。首先，它将告知利什曼原虫细胞周期的基本生物学和分子控制，允许与其他动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技术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，更好地了解它是如何工作的是利用这些好处的必要条件。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.