Microfluidic Spatial Isolation and Cytokine Profiling of Human Immune Cells

基於微流體的人體免疫細胞空間維度篩選及其細胞因子檢測

Student thesis: Doctoral Thesis

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Award date4 Mar 2021

Abstract

Immune system disease which disrupt the immune status of immune cells can cause fatal healthy problems. Leukocytes play a key role in both innate and adaptive immune responses, yet promising prognosis and diagnosis on the immune systems are very challenging due to the multiplex, heterogeneous and dynamic responses and cell-cell communication. Meanwhile, it is worth noting that many studies have reported that host-associated microbial communities played as a key role in healthy and disease states. However, lack of effective bacterial single-cell isolation techniques hindered to deeply understand interaction between effect contributed from independent bacteria in the whole communities and pathology. Therefore, there is an urgent need to develop a novel method to achieve efficient single cell/bacteria isolation and analysis at single cell level for immune system disease study. Moreover, characterization of different immune subtypes on their cytokine secretion during a sophisticate immune response can provide an important insight on mechanisms of immune diseases. However, dynamic quantitatively measurement of cytokine secretion from different isolated single-cells still remain challenge due to different immune cell with distinct behaviors, while they are being co-cultured in the same chamber.

For selective immune cell isolation, we firstly developed a microstructured device, which consists of antibody-coated micropillars and micro-sieve arrays, for isolating a target immune cell subtype from bovine blood samples. The focusing micropillars can guide immune cells flowing to the subsequent micro-sieves based on deterministic lateral shifts of the cells. Arrangement of these microstructures is characterized and configured for the maximal cell capture rate. Surface modification with a selected antibody offers selective cell capture in the micro-sieves based on the antigen-antibody reaction. We prepare a cell mixture of human CD14-expressing leukemia cells (THP-1) and epithelial cells (MDA-MB-231) in diluted blood to characterize the cell isolation operation, with a selective cell isolation yield of >80 %, cell purity of ~100 % and cell viability of >93 %. Together, this microstructured strategy can achieve high-yield selective isolation of immune cells from blood samples and support downstream genetic and biochemical cell analyses, contributing to a broad range of medical diagnosis of immune diseases.

We also presented a microfluidic based device for dynamic profiling of cytokine secretion at single cell level. This device is consisting of three modules: single cell isolation module, single functional beads isolation module and cytokine diffusion chamber which coordinate together to isolate target cell subtype into a specific position and achieve cytokine detection. This device could isolate cell and functional microbeads with high efficiency (~85%). We adopt THP-1 cells and M0, M1 macrophage as the sample, and we successfully stimulated isolated cells and quantitively measure cytokine secretion from target cell based on calculation. Therefore, we believe this studied well microfluidic based platform can provide a potential application in biology analysis clinical treatment, and biomedical engineering.

Finally, we proposed a droplet-based microfluidic integrated with sequential micro-sieve array for bacterial single-cell isolation and selective extraction. Stable monodisperse aqueous droplet with homogenous diameter could be generated in oil phase at flow focusing nozzle for encapsulating the single-bacterial sample. The oil flush guided the droplet to the single-row of micro-sieve array for sequential capturing with high efficiency (~95%). The selective droplet could be extracted by side flow for further analysis. We investigated the droplet generation procedure in the device and discussed on capturing behavior around micro-sieve. In addition, we isolated bacteria from human skin sample and the DNA inside was tested by polymerase chain reaction (PCR) and DNA sequencing technique. From these above conclusions, we believe this isolation strategy provide a potential application in microbiome and pathology researches.