Culturing And Identification Of Different Age Rat Retinal Stem Cells In Vitro

With the development of science and technology,corneal and lens diseases can be cured.But the congenital,injured and diabetes retina diseases can not be cured, further more the rate of blindness caused by the above reasons rose obviously.There is a growing concern about the stem cell research, because it gives hope for the treatment of retinal incurable injury. Associated with retinal disease stem cells involve in embryonic stem cells and adult stem cells. Retinal stem cells have many advantages of curing retinal diseases, such as having organization homology, almost no rejection, having no ethics dispute and etc. This experiment discusses the characteristics of different age Wistar rats, establishes the isolation, cultivation and identification methods in vitro, through training retinal stem cells of different age rat . As well as this experiment will be useful for the study of mechanism and potential of their differentiation. Above all, the experiment establishes a theoretical foundation for the treatment of retinal diseases.The choosed experimental animals' age are 18 days of pregnancy, 23 hours of birth, 7 days, 13 days, 21 days and 2 month of birth. Organizations are selected from the ciliary body (Containing pigment layer), which are cultured in L-DMEM/F12 medium without serum, but contains the growth factor. At the same time, isolating after birth rats' retinal tissue are cultured in the same medium, that are negative contrast. The subject of positive contrast group is rats' fetal brain tissues , cultured conditions are same as above.The retina of embryo mouse is digested as many cells, round, different in size. After 24 hours, the number of cells reduces. After 48 hours, there is some cell masses, which contains about 10 cells with strong refractive index, and as a ball. Shaking plate, the cell masses detaches from the bottom. After 4 or 5 days, the masses increase, and big cell messes appeared. To 6 or 7 days , the number of masses reach peak. Cultured 5 to 7 days , the masses pass on from generation to generation. After subculture, the cell can re-form cell clusters. The third generation is hard to re-form cell masses.For the after birth rats, the ciliary body can grow to cell masses, while the number of masses become smaller than embryo ones. The retinal cell can not grow in this kind of medium. The brain cell can grow to neural stem cells, which shape is the as same as the cell from ciliary body.Retinal stem cells of primary and after generations were nestin antigen staining, microscopy can be seen in different stages of different cell culture patterns, and positive immunofluorescence staining. Negative contrast group shows no fluorescence. And antigen expression after passage through the separation has no significant attenuation.Cells fetal rat brain tissue formed antigen nestin staining for bright fluorescence , and microscope shows in different stages of cell in different patterns. Negative contrast group showes no fluorescence.Cells culture in the switch L-DMEM/F12 of 10% FBS culture medium, after 48 hours, cells began to "budding", grow a small protuberance, 6 ~ 7 days, shows a typical morphology of neurons.Through different age rat retinal stem cells isolation, cultivation and identification , conclusions as follows: Embryo can be cultured rat retinal precursor cells of the retina, and retinal phenotype similar to stem cells.The age from after birth to the adult stage in rat ciliary body have neural stem cells of self-renewal and multi-differentiation potential , retinal stem cells isolated from birth within 13 days rat ciliary body are more easily culturing in vitro. Retinal of after birth in rat can not be found stem cells in vitro.