Cell Demographics from Full Thickness Retinal Explant Growth on Micropatterned Surfaces

Abstract

Abstract: : Purpose: The effectiveness of retinal prosthetics is limited by the distance between stimulating electrodes and retinal cells. Our goal was to see whether neurites could be directed to grow in a controlled fashion out of full thickness retinal explants, and to identify the neurite-producing cells. Methods: Full thickness retinal explants were harvested from P7 Spraque-Dawley rats. The explants were cut into 1-mm squares and cultured on a larger micropatterned surface of either thermonox, polystyrene, polyacrylic acid and polyacrylamide. Prior to seeding, substrates were coated with poly-D-lysine for 2 hours, then patterned with laminin at a concentration of 200 ug/mL using microcontact printing. Poly (dimethylsiloxane) stamps used for microcontact printing were formed from photoresist-coated silicon substrates (molds). These molds were fabricated using standard photolithographic techniques. The explants were cultured at 37 °C and 6.5% CO₂ in 1 mL of serum-free medium (supplemented with growth factors Neurobasal-A) for 3 weeks. Antibodies to Thy 1.1, Neurofilament 200 and GFAP (glial fibrillary acidic protein) were used to visualize and identify different cell types. Results: The neurotrophic growth factors were found to induce neurite outgrowth from the edges of full thickness retinal explants. When the growing surface was micropatterned with laminin, single retinal cell neurites followed the micropatterns and thus could be directed to specific electrode sites. A variety of materials including thermonox, polystyrene, polyacrylic acid and polyacrylimide surfaces could be used as substrates for micropatterning. Immunohistochemical studies indicated that the neurites we observed were of retinal ganglion cell origin although they often carried some glial cell tissue in accompaniment. Conclusions: The ability to pattern neurites growing from a full-thickness retinal explant allows for the possibility of directed growth towards an electrode for specific, addressable stimulation of retinal neurons by a prosthetic device.

Keywords: retinal connections, networks, circuitry • retinal culture