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Wyndham More Batchelor, Galina Dvoriantchikova, Dmitry V Ivanov, Vincent Moy, Noel M Ziebarth; Custom Atomic Force Microscope Lithography System to Create Guidance Cues for Retinal Ganglion Cell Axon Growth. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2519.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cells (RGCs) are the only retinal neurons that send their axons to the visual cortex of the brain. Since axon damage and RGC loss caused by diseases and conditions lead to vision loss, researchers are creating tissue engineered RGC replacement therapies. However, guiding RGC axons from the retina to the brain still is a major challenge. We developed a custom lithography system based on atomic force microscopy techniques to create axon guidance cues; we then applied our system to create linear topological features in a cell culture dish to determine if these features influence the directionality of RGC axon growth.
A custom-developed atomic force microscope adapted for lithography was used for these experiments. A silicon nitride cantilever with a pyramidal tip (Veeco, MLCT, k = .01N/m) was lowered onto the surface of a 35mm cell culture dish (Falcon 353001) until it was in contact. Grooves were created by indenting the dish with the cantilever tip by lowering it approximately 2µm or 14µm using either the piezoelectric actuator or manual actuators. Once in contact, the x-y stage that controls dish movement was manually translated to make grooves 4 mm in length. Following lithography, dishes were rinsed with sterile water and then sterilized with antiseptic UVA light. Primary RGCs were isolated from mouse retinas according to the two-step immunopanning protocol and plated on the dish close to the groove in serum-free neurobasal/B27 media (Thermo Fisher Scientific) and grown at 37°C, 5% CO2. The dishes with RGCs were imaged 5 days later using light microscopy to observe axon growth, and analyzed using ImageJ software.
Our custom system successfully created well-defined topological features on the cell culture dishes. We observed living RGCs and extensive neurite growth following incubation. RGCs extended axons over topological features, but axon directionality did not appear to be influenced by either the 2mm or 14mm deep grooves created in the surface.
AFM lithography is a versatile method of creating very well defined, high resolution features on a substrate of interest. This method has the potential to be expanded upon by adding chemical patterning modalities and applied to tissue engineering the optic nerve to better guide axons to the optic nerve head.
This is a 2020 ARVO Annual Meeting abstract.
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