![]() Because most of immune cells are anchorage independent, they can be aspirated after completion of the experiment, leaving behind cytokine spots that correlate with single cells. This approach is based on seeding and activating immune cells in micro-titer plates coated with anti-cytokine Abs. ELISpot on the other hand can be used for detecting cytokines released by live cells. 6, 7 FC-based analysis relies on detection of cytokines accumulated inside fixed and permeabilized cells and therefore has limited options for time-course experiments or post-detection processing of cells. In immunology, single cell level cytokine detection is traditionally performed using either intra-cytoplasmic cytokine staining (ICS) coupled with polychromatic flow cytometry (FC) or enzyme-linked immunospot (ELISpot) assay. The functional heterogeneity of T-cells necessitates development of tools that help determine cytokine production of individual cells. In particular, Th1 phenotype of CD4 T-cells –characterized by production of IFN-γ – correlates with vigorous immune response and protection against infections and is also monitored to determine vaccine efficacy. 2, 3 Specific T-helper subtypes play an important role in the immune response. 1 Importantly, CD4 T-cells of identical morphology and surface markers are further categorized into T-helper 1 (Th1), Th2, Th17 solely based on the types of cytokines they secrete. A particular T-cell subset, T-helper cells (CD4 T-cells), regulate immune cell recruitment and proliferation through the production of a wide variety of cytokines. T-cells play a critical role in the immune response against viral and bacterial infections. The device and process presented here will be expanded in the future to enable multi-parametric functional analysis of immune cells organized into high density single cell arrays. ![]() Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-γ cytokine signal co-localized with specific T-cells. Introduction of red blood cell (RBC) depleted whole human blood followed by controlled washing led to the isolation of individual CD4 T-cells within PEG microwells. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and non-fouling walls. To define sites for single cell attachment, PEG hydrogel microwells (20 μm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-γ and anti-CD4 antibodies (Abs). The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. In the present study we employed microfabricated surfaces to capture T-cells from minimally processed human blood, arrange these cells into a single cell array and then detect interferon (IFN)-γ released from individual cells. The cytokine production by leukocytes correlates with body’s ability to mount an immune response and therefore has high diagnostic value.
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