Cell Characterization and Separation Using Microfluidics for Biomedical Applications

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.