Methods and systems for extended in vitro culture of neuronal cells

Abstract

A cell culture system related to extended in vitro culture of mature neuronal cells and methods for preparing the cell culture system are provided. In a preferred embodiment the invention provides a cell culture system comprising a mixture of mature neuronal retinal cells and cells isolated from a ciliary body. Methods for identifying bioactive agents that alter neurodegeneration of neuronal retinal cells are also provided.

1 . A cell culture system comprising a mixture of mature neuronal cells and cells isolated from a ciliary body. 
     
     
         2 . The cell culture system according to  claim 1  wherein the mature neuronal cells comprise mature retinal neuronal cells. 
     
     
         3 . The cell culture system according to  claim 2  wherein the mature retinal neuronal cells are selected from the group consisting of bipolar cells, horizontal cells, amacrine cells, ganglion cells, and photoreceptor cells. 
     
     
         4 . A retinal cell culture system comprising a mixture of mature retinal neuronal cells and cells isolated from a ciliary body. 
     
     
         5 . The retinal cell culture system according to  claim 4  wherein the mature retinal neuronal cells are selected from the group consisting of a bipolar cell, a horizontal cell, an amacrine cell, a ganglion cell, and a photoreceptor. 
     
     
         6 . A retinal cell culture system comprising a mixture of mature retinal neuronal cells and cells isolated from a ciliary body, wherein the mature retinal neuronal cells are selected from the group consisting of bipolar cells, horizontal cells, amacrine cells, ganglion cells, and photoreceptor cells. 
     
     
         7 . A retinal cell culture system comprising a mixture of (i) mature retinal neuronal cells; (ii) cells isolated from a ciliary body; and (iii) embryonic retinal cells. 
     
     
         8 . The cell culture system of  claim 7  wherein the embryonic retinal cells comprise retinal stem cells. 
     
     
         9 . The cell culture system of  claim 7  wherein the embryonic retinal cells comprise embryonic retinal progenitor cells. 
     
     
         10 . The cell culture system of  claim 7  wherein the mature retinal neuronal cells are selected from the group consisting of bipolar cells, horizontal cells, amacrine cells, ganglion cells, and photoreceptor cells. 
     
     
         11 . A method for producing a retinal cell culture system comprising co-culturing a mature retinal neuronal cell and a cell isolated from a ciliary body. 
     
     
         12 . A method for enhancing survival of a mature retinal neuronal cell in vitro comprising co-culturing a mature retinal neuronal cell and a cell isolated from a ciliary body. 
     
     
         13 . The method according to either  claim 11  or  claim 12  comprising co-culturing (i) a mature retinal neuronal cell; (ii) a cell isolated from a ciliary body; and (iii) an embryonic retinal cell. 
     
     
         14 . The method according to  claim 13  wherein the embryonic retinal cell is selected from the group consisting of a retinal stem cell and an embryonic retinal progenitor cell. 
     
     
         15 . A method for identifying a bioactive agent that is capable of enhancing survival of a neuronal cell, comprising (i) contacting a candidate agent with a cell culture system according to any one of  claims 1 - 10 , under conditions and for a time sufficient to permit interaction between a neuronal cell of the cell culture system and the candidate agent; and (ii) comparing survival of a neuronal cell of the cell culture system in the presence of the candidate agent with survival of a neuronal cell of the cell culture system in the absence of the candidate agent, and therefrom identifying a bioactive agent that is capable of enhancing survival of the neuronal cell. 
     
     
         16 . A method for identifying a bioactive agent that is capable of inhibiting neurodegeneration of a neuronal cell comprising (i) contacting a bioactive agent with a cell culture system according to any one of  claims 1 - 10 , under conditions and for a time sufficient to permit interaction between a neuronal cell of the cell culture system and the candidate agent; and (ii) comparing structure of a neuronal cell of the cell culture system in the presence of the bioactive agent with structure of a neuronal cell of the cell culture system in the absence of the bioactive agent, and therefrom identifying a bioactive agent that is capable of inhibiting neurodegeneration of the neuronal cell. 
     
     
         17 . A method for identifying a bioactive agent that is capable of treating a retinal disease comprising contacting a bioactive agent with a cell culture system according to any one of  claims 1 - 10 , under conditions and for a time sufficient to permit interaction between a neuronal cell of the cell culture system and the candidate agent; and (ii) comparing neurodegeneration of a neuronal cell of the cell culture system in the presence of the bioactive agent with neurodegeneration of a neuronal cell of the cell culture system in the absence of the bioactive agent, and therefrom identifying a bioactive agent that is capable of treating a retinal disease. 
     
     
         18 . The method according to any one of  claims 15 - 17 , wherein the neuronal cell is a retinal neuronal cell. 
     
     
         19 . The method of  claim 17  wherein the retinal disease is selected from the group consisting of macular degeneration, glaucoma, diabetic retinopathy, retinal detachment, retinal blood vessel occlusion, retinitis pigmentosa, and a retinal disorder associated with Alzheimer's disease. 
     
     
         20 . A method for treating a retinal disease comprising introducing isolated retinal stem cells into retinal tissue of a subject in need thereof. 
     
     
         21 . The method of  claim 20  wherein the retinal disease is selected from the group consisting of macular degeneration, glaucoma, diabetic retinopathy, retinal detachment, retinal blood vessel occlusion, retinitis pigmentosa, and a retinal disorder associated with Alzheimer's disease.