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Katsuhiko Shinomiya, Satoshi Kawasaki, Keita Aoi, Koji Kitazawa, Shigeru Kinoshita; A New Method for the Efficient and Less-Stress Dissociation of Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):540.
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© ARVO (1962-2015); The Authors (2016-present)
For the primary culture of corneal epithelial cells (CECs) from human and animal ocular tissue, the cells should be enzymatically separated and dissociated from the tissue via the use of proteases such as dispase or trypsin. However, it is well known that protease treatments have some cytotoxic effects and may cause cell damage. In this present study, we report a new technique for the enzymatic dissociation of CECs which allows for lower cytotoxicity and a higher yield of cells.
The central region (8.75-mm diameter) of the cornea was trephined from the eye of a Japanese white rabbit and treated with dispase for 1 hour at 37°C (Method A) or overnight at 4°C (Method B). While being observed under a microscope, the corneal epithelium was then carefully separated from the underlying stroma. The separated corneal epithelium was further dissociated by the use of a trypsin-like protease. The histology of the separated corneal epithelial sheet was assessed by hematoxylin-eosin staining. The dissociated cells were counted and observed under a phase contrast light microscope for the assessment of their cell morphology. For the assessment of apoptotic status, the dissociated cells were subjected to a commercial chemiluminescence-based apoptosis assay.
The average number of CECs obtained by the Method B was 1.38±0.15×106 cells per corneal button, which was significantly larger than that obtained by the Method A (6.75±0.46×105 cells per corneal button). Compared to the Method A, the Method B resulted in a significantly lower number of apoptotic cells. A significantly larger number of large cell aggregates were observed in the cell suspension of the Method A compared with that of the Method B. Significantly severe histological damage was found in the corneal epithelial sheets obtained by the Method A compared to those obtained by the Method B.
The findings of this study show that a low-temperature dispase treatment method produced a higher yield of dissociated CECs with lower cytotoxicity. This method should prove useful in research involving CEC cultures, as well as corneal epithelium-related regenerative medicine.
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