Study On Culturing Corneal Epithelial Cells, Keratocytes And Endothelial Cells In Gravity And Simulated Microgravity

Objective: (1) To research the characteristics of corneal epithelial cells, keratocytes and endothelial cells of rabbit which were cultured in vitro, and to lay a foundation for tissue engineering of cornea. (2) To observe the growing properties of three corneal cell types cultured on amniotic membrane in order to further study on using amniotic membrane as biomaterials for layering tissue engineered cornea. (3) To explore corneal epithelial cells, endothelial cells and keratocytes of rabbit cultured for three-dimensional modeling in simulated microgravity.Methods: (1) Rabbit corneal epithelial cells, keratocytes and endothelial cells were cultured by using tissue inoculation or digestion methods and the cells were subcultured after they became confluent. The growing characteristics were observed every day. (2) Rabbit limbal corneal epithelial cells, keratocytes and corneal endothelial cells were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Scanning electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. (3) Rabbit corneal epithelial cells, keratocytes and endothelial cells, attached to the bovine corneal stroma as carrier or not, were added into rotating wall vessel bioreactor to culture in simulated microgravity. The proliferation of cells was assessed by CCK-8 assay, and the cultivated cells were observed under inverted phase contrast microscope and scanning electron microscope.Results: (1) The epithelial cells, keratocytes and endothelial cells adhered and grew within 24-48 hours and formed a confluent monolayer in 6-9 days. The shape of the primary isolated cells was typical, and the subcultural cells still maintained the characteristics of the primary cells. The cells grew very well and could be cultured for a long term. (2) Three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints whichcould form networks. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. (3) Three rabbit corneal cell types were able to proliferate in simulated microgravity. The epithelial cells showed sphere morphology with the tight intercellular joints and were gathered into a mass. CCK-8 assay showed the keratocytes and corneal endothelial cells proliferated more in the simulated microgravity than in the normal gravity. The keratocytes and corneal endothelial cells cultured in simulated microgravity showed the natural morphology.Conclusions: (1) The research established a simple, highly efficient method for culture of rabbit corneal epithelial cells, keratocytes and endothelial cells in vitro. (2) Amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea. (3) The rotary cell culture system is suitable to develop rabbit corneal keratocytes culturing for three-dimensional modeling. Corneal endothelium can be reconstructed in simulated microgravity by cultivating in the rotating wall vessel bioreactor. It's necessary to do more research on the effect of simulated microgravity on the cultivation of rabbit corneal epithelial cells...