Quantitative evaluation system for tissue-engineered corneal epithelial cell sheets using image-based technology

Background: Tissue-engineered corneal epithelial cell sheets have been applied for treating ocular surface diseases. It is essential to establish a standardized validation system for fabricated cell sheets before transplantation to spread the therapy in the world. It has already been reported that a validation system for tissue-engineered corneal epithelial cell sheets was developed, in which several sliced frozen sections of sheet were fluorescently stained and the each was subjectively quantitated under a microscope with naked eye. In this study, we attempted a platform for an evaluation system which can be more simplified in preparation steps and more objective in quantification by automatically observing wider area of sample areas. Methods: Human limbal epithelial cells isolated from corneoscleral rims of cadaveric donors (n=4) were cultured with 3T3 feeder layer cells on a temperature-responsive culture insert for two different culture periods, for day 15 and 28. The fabricated cell sheets, without slicing them, were fluorescently stained with five antibodies and with DAPI for nuclear staining, and then a wider area of the sheets were automatically scanned by a confocal microscope and quantitatively analyzed using a 2-D image processing technique with combination of two spatially-cropped images, lower and upper, which are created by dividing stacked image at the center of its z-direction. Results: The cell sheets harvested on day 15 consisted of 4-6 layers of cells, the number of which in the lower was about 1.5 to 2.0 times larger, compared to that in the upper while on day 28 they had only 2-3 layers with the decreased number of cells by about 50% in the lower. The differences in the protein expressions the culture periods brought about were successfully quantitated by image processing technique as well. On day 15, ZO-1, AE5 and MUC16 were predominantly expressed in the upper while the expression of panCK was seen in every cell on the sheet. On day 28, the expression levels of ZO-1, AE5, MUC16 and panCK were decreased in the both layers with some unique features. Cells expressing p63 were predominantly observed in the basal layer of the sheets on day 15, but the level was decreased on day 28 as well. Conclusions: The combinational image-based technology could be one of the powerful tools in establishing a standardized validation system not only for corneal treatment but for some other tissue-engineered ones.