关键词: 组织培养, 方法, 三维培养

Abstract: Objective To establish a new culture system for retinal ganglion cells (RGCs). Methods Four male Wistar rats aged 10 weeks, retina was isolated from the choroid, the pigment epithelium and adherent vitreous. The retinal explants from mid-peripheral retinas were dissected into 16 pieces (approximately 0.5 mm×0.5 mm square). Four pieces of retinal tissues were embedded in the type I collagen gel with the nerve fiber layer downward on plastic dish. Each dish was pre-coated with 10 mg•L^-1 poly-L-lysine. The explants were cultured in serum-free MEM and maintained at 37℃ in an incubator under a 5% CO₂/95% air atmosphere. The number of outgrowing neural protuberances was counted under phase contrast optics using inverted microscope. Immunohistochemical markers, monoclonal antibodies against neurofilament and Thy 1.1 were used to identify the nature of the outgrowing processes. Results Using the three-dimensional culture, the retinal explants were stably embedded just below the surface of the gel, and they could be cultured for 2 weeks or more. The outgrowing neural protuberances could elongate more freely into gel in the three-dimensional culture than in the two-dimensional one. Conclusion RGCs can be cultured sucecessfully in the three-dimensional culture.

Key words: tissue culture, methods, three-dimensional culture