Growth Pattern of Sirc Rabbit Corneal Cells in Microwell Inserts

Abstract The SIRC rabbit corneal cell line (ATCC CCL 60) has been used in a number of in vitro studies. However, the growth pattern of SIRC rabbit corneal cells on microwell inserts has not been established. Microwell inserts were seeded with SIRC rabbit corneal cells, suspended in growth medium. Cultures were maintained in a humidified incubator (5% CO237°C) for 21 days, with the growth medium replaced in the wells and inserts every seventh day of culture. Light microscopy was performed on hematoxylin and eosin-stained cross sections. The SIRC rabbit corneal cells formed multiple epithelioid cell layers. Maximum cell layer growth occurred at day 10 (4.54 cell layers ± 0.32). Although decreased slightly, the number of cell layers remained stable with an average of more than 4 cell layers observed for days 12-14. On the 21st day of culture, the average number of cell layers decreased to 3.87 (± 0.09) and exfoliation of the surface cells was evident. Microscopy of the cross sections indicates that new cell layers were formed closest to the microwell insert. The cells found next to the filter appeared small and had a lower cytoplasm to nucleus ratio when compared to those cells farthest from the filter. A single inoculation of SIRC rabbit corneal cells resulted in the formation of multiple epithelioid cell layers, with the number of layers increasing with culture time. Cell growth and aging appeared to be similar to that seen in the in vivo environment, from the basal to apical layer. The present study shows promise as an in vitro model for use in the assessment of corneal permeability of chemical substances.