Optical-quality surface that imparts spatial control of macrophage fusion
Inventors
Faust, James • Ugarova, Tatiana • Ros, Robert • Christenson, Wayne • Doudrick, Kyle
Assignees
University of Notre Dame • National Institutes of Health NIH • Arizona State University Downtown Phoenix campus
Publication Number
US-11591574-B2
Publication Date
2023-02-28
Expiration Date
2037-09-08
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Abstract
Methods to form a surface coating and surface pattern, which are based on adsorption of hydrocarbon chains that can be used with imaging optics to visualize macrophage fusion and multinucleated giant cell formation with living specimens are described.
Core Innovation
The invention concerns an optical-quality glass surface with a hydrocarbon-based coating, optionally possessing micropatterns, that facilitates high rates of macrophage fusion. This surface is compatible with microscope techniques, especially advanced light microscopy, allowing for visualization of macrophage fusion and multinucleated giant cell (MGC) formation in living specimens. The surface coating is based on the adsorption of hydrocarbon chains, such as oleamide, paraffin wax, or petrolatum, which are non-covalently coupled to the optically clear glass substrate. Micropatterns, such as a grid pattern, can be introduced to impart precise spatial control of cell fusion.
Prior to this invention, there was a deficiency in the ability to visualize macrophage fusion in vitro using living specimens, due to the absence of surfaces that simultaneously promote macrophage fusion and maintain optical properties suitable for advanced imaging techniques. Common fusogenic surfaces such as plastics were not optimal for high-resolution imaging, while optical-quality glass did not support adequate macrophage fusion. The disclosed surface overcomes these limitations by supporting robust macrophage fusion and enabling detailed live imaging under a microscope.
The methods described include treating the glass surface with vacuum gas plasma and adsorbing it with a solution containing selected hydrocarbons, creating a hydrophobic and fusogenic surface. Micropatterning can be achieved via stamping or masking techniques, resulting in regions with controlled roughness and topography conducive to directing the spatial occurrence of cell fusion. This approach allows not only the visualization of macrophage fusion as it occurs but also the study of the kinetics and mechanisms underlying MGC formation, as the process can be precisely observed in real time using advanced microscopy modalities.
Claims Coverage
The patent presents two independent inventive features describing methods for adhering cells to a coated glass surface and visualizing macrophage fusion using microscopy. These features focus on hydrocarbon-coated, micropatterned glass surfaces that promote and reveal macrophage fusion.
Method of adhering a monocyte or macrophage to a hydrocarbon-coated, micropatterned glass surface
This method comprises: - Coating a glass surface with a hydrocarbon that is non-covalently coupled and treating the glass with vacuum gas plasma. - Providing a hydrocarbon coating with a micropattern, specifically a grid pattern composed of an array of hydrocarbon material raised from the surface to a peak of about 10 nm. - Contacting a monocyte or macrophage with this coated surface under conditions suitable for cell fusion. The invention covers methods of producing the micropattern using stamps or grids and specifies hydrocarbon choices including oleamide, paraffin wax, and petrolatum.
Method for visualizing macrophage fusion in living specimens with microscopy on hydrocarbon-coated, micropatterned glass
This method includes: - Contacting a monocyte or macrophage to a glass surface coated with a hydrocarbon suitable for light microscopy, where the surface has a micropattern (grid) comprising an array of hydrocarbon coating material raised to a peak of about 10 nm. - The hydrocarbon is selected from oleamide, paraffin wax, and petrolatum. - Visualizing macrophage fusion using microscopy. The inventive features also include applying interleukin-4 to induce cell fusion, optionally applying macrophage-1 antigen (Mac-1), and providing conditions to promote multinucleated giant cell formation. The method specifies the use of borosilicate glass with a particular thickness.
The patent claims methods that enable spatially controlled macrophage fusion on optically suitable glass surfaces via non-covalently coupled hydrocarbon coatings with micropatterns, providing means to induce and directly visualize cell fusion using advanced microscopy.
Stated Advantages
Allows robust macrophage fusion on optical-quality glass surfaces while maintaining compatibility with advanced microscopy techniques.
Enables direct, real-time visualization of macrophage fusion and multinucleated giant cell formation in living specimens.
Micropatterned surfaces impart precise spatial and temporal control over the site and timing of macrophage fusion events.
Hydrocarbon-coated surfaces are compatible with high-resolution imaging modalities, such as total internal reflection fluorescence microscopy and super-resolution microscopy.
Overcomes limitations of traditional surfaces that either lack fusogenicity or are not optically suitable for advanced imaging.
Documented Applications
Visualization of macrophage fusion and multinucleated giant cell formation in living specimens via light microscopy.
Direct, live imaging of cell fusion processes for studying cellular and molecular mechanisms underlying macrophage fusion.
Use of micropatterned surfaces to achieve spatiotemporal control for studies of sequential fusion events and MGC formation kinetics.
Application in advanced microscopy techniques such as total internal reflection fluorescence microscopy and direct stochastic optical reconstruction microscopy (dSTORM).
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