les were washed again. The cells were examined by fluorescence microscope. Scanning electron microscopy. Scanning electron microscopy was used to observe the ultrastructure of the surface of the cells and carriers or the growing morphology of keratocytes. Samples were fixed in 2.5% glutaraldehyde, washed three times for 30 min each time in 0.1 M PBS, and then postfixed in 1% osmium tetroxide for 30 min. Samples were washed three times again in PBS before passing through a graded series of alcohol. After three 5-min changes of 100% ethanol, the samples were then transferred to isoamyl acetate for 30 min, critical point dried, coated with gold, and mounted for viewing in the JSM-T300 SEM. Statistical analysis. The values were expressed as means 6 SD from three to six samples. Statistical analyses were carried out using Student’s t test and a one-way analysis of variance. Results of p,0.05 were considered statistically significant. Results The observation of decellularized bovine cornea H&E staining showed that cells were eliminated in our decellularizated bovine cornea, while there were many keratocytes in the normal bovine corneal stroma. SEM evaluation indicated the rough surfaces of bovine acellular stromal lamella, which were composed of a series of fibers and shallow pores at the lower magnification as well as collagen fibers arrayed regularly parallel and MedChemExpress MK-886 formed microporous structure at the higher magnifications. The cross section of acellular stroma had rough surface constituted by abundant lamellar structure. This result revealed that the cells of bovine cornea were removed by using our shortterm chemical-frozen decellularization.Effects of VPA, VC and RCCS on Rabbit Keratocytes The effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes The proliferations of keratocytes on the decellularizated bovine cornea or plastic were significantly promoted when supplemented with 1 mM VPA and 50 ug/ml VC based on CCK-8 assay. The cell-cycle entrance of keratocytes treated with 1 mM VPA and 50 ug/ml VC was significantly higher than keratocytes of control group without VPA and VC. The percentage of cells entering the S phase and G2/M phase in the VPA and VC group and control group were % and % respectively. Annexin V and PI were analyzed by flow cytometry to detect apoptosis in cultured keratocytes. Keratocytes challenged with H2O2 showed % apoptotic cells, whereas keratocytes added 1 mM VPA and 50 ug/ml VC under the same challenge displayed % apoptotic cells. The result showed that VPA and VC were able to reduce the ratio of apoptotic keratocytes challenged with H2O2. smaller ellipse shape and formed reticular structure at day 1. Discriminatively, at day 4 of SMG culture, a large number of keratocytes interconnected and formed three-dimensional aggregates, which was a distinctive phenomenon in SMG experiment group. Further, the spherical aggregation and proliferation of keratocytes became larger and more obvious at day 7 of SMG culture. The observation of keratocytes by light microscopic evaluation In the presence of 10% FBS, almost all keratocytes on plastic in static culture without VPA and VC and with VPA and VC showed spindle shape, and rare irregularly interconnected or unconnected with each other. However, also 10% FBS in existence, keratocytes on the carriers of acellular bovine cornea in static culture with VPA and VC well adhered to carriers and interconnected to form reticular structure at day 1 of