成熟视网膜神经节细胞三维培养

Establishment of new culture system for retinal ganglion cells

目的 :建立成熟视网膜神经节细胞的三维培养模型。方法 :将边长 5 0 0 μm的方形网膜片神经纤维层向下浸入 1 2孔培养板的胶原溶液层中 ,胶原溶液凝固后 ,培养孔内加入无血清 MEM培养液。相差显微镜观察生长情况。应用羊抗鼠 FITC- Thy- 1 .1单克隆抗体鉴定培养的神经节细胞突起。结果 :视网膜神经节细胞突起在凝胶中呈螺旋迂曲生长 ,可存活 2周以上。免疫组化荧光显色神经节细胞突起呈阳性反应。结论 :应用凝胶做支持物对成熟视网膜神经节细胞进行三维原代培养 ,是一种成功的。

ObjectiveTo establish a new culture system for retinal ganglion cells (RGCs). MethodsFour 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% CO 2/95% 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. ResultsUsing 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.ConclusionRGCs can be cultured sucecessfully in the three dimensional culture.