Microfluidic Single-cell Isolation and Measurement for Spatial Profiling of Cytokines Secreted from Different Immune Cell Subtypes

Immune diseases involve disruption to immune cell behaviors and cause fatal health problems. Leukocytes are important in both innate and adaptive immune responses, yet promising prognosis and diagnosis on the immune responses are very challenging, mainly hindered by the multiplex, heterogeneous and dynamic responses and cell-cell communications (e.g. cytokine secretion) among different immune cell subtypes in blood. Characterization of different immune subtypes on their cytokine secretion during a systematic immune response can provide important insights on mechanisms of immune diseases as well as effective treatment strategies. In particular, cell isolation from blood is a key step for diagnosing immune diseases. Despite demands on efficiency and purity of cell isolation, placement of the isolated cells at defined positions in a co-cultured environment is equally critical. Cell-cell communication should be maintained among the isolated cells such that the in vitro stimulated cytokine secretions from those isolated cells are more representative to in vivo immune responses. Additionally, it is technically very challenging to quantitatively measure the cytokine secretions from different isolated single-cells, which should belong to different immune cell types with distinct behaviors, while they are being co-cultured in the same microchamber. Considering also involvement of multiple cytokine types in an immune response, it is necessary to establish a multiplex cytokine quantification technique for different local sites over the co-culturing microchamber. This proposed research aims at developing a novel microfluidic ‘immunophenotyping’ device for determining cytokine secretions from different leukocyte subtypes during immune responses of an immune cell mixture. For the single-cell isolation, we plan to apply arrays of microstructures (micropillars and micro-sieves) to displace and trap the cells at defined positions in a co-culture region. Dimensions of the microstructures should match the immune cells; and arrangement of these microstructures is configured for the maximal cell capture rate. Further, the spatial cytokine concentration profile is measured by an array of detection microbeads for different cytokine molecules pre-captured over the co-culture region. The cell subtypes can be subsequently identified based on their surface protein expressions. Hence, we can convert the measured spatial profiles of cytokines and cell types to the cytokine secretion of different leukocyte subtypes during a target immune response. Altogether, the cell subtype-specific multiplex cytokine secretions quantified by the proposed microfluidic device are a unique signature that characterizes patients’ immunophenotypes more precisely. This can lead to better prognosis and diagnosis of immune diseases and disorders, allowing for personalized medications and effective early-stage treatment.