Detecting cytokine release from single T-cells.
The cytokine production by leukocytes correlates with body's ability to mount an immune response and therefore has high diagnostic value. 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)-gamma released from individual cells. The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-gamma and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 microm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and nonfouling walls. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. 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. Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-gamma cytokine signal colocalized with specific T-cells. The device and process presented here will be expanded in the future to enable multiparametric functional analysis of immune cells organized into high density single cell arrays.
Zhu
He
H
Biomedical Engineering, University of California, Davis, USA.
Stybayeva
Gulnaz
G
Silangcruz
Jaime
J
Yan
Jun
J
Ramanculov
Erlan
E
Dandekar
Satya
S
George
Michael D
MD
Revzin
Alexander
A
D055910:Antibodies, Immobilized / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D007371:Interferon-gamma / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D007371:Interferon-gamma