Development of Microfluidic Platform for Isolation, Incubation and Cytokine Profiling of Human Immune Cells
Student thesis: Doctoral Thesis
Related Research Unit(s)
Quantitative and dynamic analyses of immune cell secretory cytokines are essential for precise determination and characterization of the “immune phenotype” of patients with immune-related diseases in clinical diagnosis and treatment. In the past few decades, multiple immunoassay techniques were developed to satisfy the requirement of testing and some of them were chosen as gold standard methods. These traditional methods require a series of steps involving complicated diluting, blocking and washing, which would decrease the sensitivity and efficiency of the measurement. Functional microbeads have been widely applied in molecular identification and other biochemical applications in the past decade, owing to the compatibility with flow cytometry and the commercially available microbeads for a wide range of molecular identification. For inflammatory cytokine beads, a minimal sample volume of 50 μl required for single group is not suitable for sampling repetition, imposing negative limitations on other experimental conditions. Therefore, microfluidics is proposed to overcome the deficiencies of conventional methods in our research process. In the first work, we optimize the operation of an automated microbead-based microfluidic device for the reagent mixing and the dynamic cytokine detection. In particular, we adopt fluorescence microscopy for quantification of multiple microbeads in each microchamber instead of flow cytometry for a lower detection limit. In the second step, an improved mixing device, which can effectively meet the detection requirements for characterizing the rebuilt immune response in vitro, is presented. In order to achieve dynamic monitoring of cytokines, the existing design is optimized and a cell culture chamber is added for on-chip incubation. This comprehensive platform ensures that a certain amount of cell medium can be extracted into the detection chamber at any time without affecting the culture balance. It is noteworthy that immune cells include different subtypes and their cytokine level vary from one subtype to the other. Therefore, in the third step of the work, we report a new chip for single-cell isolation, and a series of antibody-coated micro-sieves is utilized to further select target cells specifically. In future work, we will improve the existing separation device by adding inlets and outlets on both sides of each row of micro-sieves, thus fulfilling the requirement of discharging untargeted cells. Different separation chambers will be connected by microchannels to further achieve continuous cell separation. The final platform will include several separation chambers and a detection unit, and then a set of automated control procedures will be programmed to realize automatic on-chip separation and immune dynamic monitoring of human immune cells. We also hope that the proposed design would provide new references for the update and development of medical equipment.