Purchase this article with an account.
N. Koizumi, Y. Sakamoto, Y. Ban, N. Hirai, Y. Ishino, J. Hamuro, S. Kinoshita; The Cultivation of Monkey Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4526.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish a method of cultivating monkey corneal endothelial cells (MCECs) into a confluent sheet on a collagen carrier. Methods: Descemet’s membrane with MCECs were obtained from 12 cynomolgus monkeys (3–11 years old) at euthanasia for other purposes. MCECs were dissociated from Descemet’s membrane after immersion in 1.2IU dispase for 1 hour. Cells obtained from each eye were divided and incubated for 1–2 weeks to confluence in 48 separate wells coated with collagen type IV. Confluent subcultures were then transferred to uncoated wells, or wells coated with collagen type I, collagen type IV, FNC coating mix, gelatin, poly–L–lysine (PLL) or poly–D–lysine (PDL). Two subcultures were cultivated on collagen type I carriers. After cultivation periods of up to 11 days on wells and up to 5 weeks on the collagen carrier, cell proliferation rates and cell morphology were assessed. Results: Primary cultures of MCECs formed confluent layers of hexagonal cells within 7–14 days for tissues obtain from seven of the twelve monkeys. Cell density ranged from 824 to 2720 cells/mm2 (mean=1806±715 cells/mm2 ), and was greater for cells from younger animals . MCECs proliferated more rapidly on wells coated with FNC coating mix, collagen type I, and collagen type IV compared to cells on uncoated plates (P<0.001). Poor proliferation was seen on PLL and PDL coated plates. MCECs reached confluence on the collagen type I carrier and achieved cell density of over 2800 cells/mm2, similar to the in vivo situation. Cell morphology on the collagen carrier was similar to normal, and cell–cell junctions were in evidence with ZO–1 tight junction expression. Conclusions: As in humans, corneal endothelial cells in the monkey do not proliferate in vivo. To date, most work with endothelial cell cultivation has dealt with rabbits, whose endothelial cells have regenerative properties. Here, we report a monkey model for the fabrication of corneal endothelial cell sheets with a view to transplantation. We also show how MCECs cultivated on collagen type I carriers form intact endothelial sheets with potential applications for tissue engineering of cornea.
This PDF is available to Subscribers Only