Purchase this article with an account.
Ayaka Kusakabe, Naoki Okumura, Hiroatsu Hirano, Shigeru Kinoshita, Noriko Koizumi; The effect of cell density on the adhesion and proliferation properties of cultivated corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2058.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We previously reported the feasibility of cultivated corneal endothelial cell (CEC) cell-injection therapy using a rabbit corneal endothelium dysfunction model (Okumura N, et al. Am J Pathol, 2012). CEC density (CD) is known to be an important index for evaluating the healthiness of corneal endothelium in the clinical setting. The purpose of this present study was to investigate the effect of cultivated CEC CD on cell adhesion and proliferation properties to establish an optimum product specification for the future clinical application of cell-injection therapy.
Rabbit CECs (RCECs) were cultured by DMEM supplemented with 10% FBS. RCECs of which CD were 2540.4±64.9 cells/mm2 were used as High-CD cell and 720.2±29.4 cells/mm2 were used as Low-CD cells for this study. Cellular morphology and expression of function-related proteins such as ZO-1 and Na+/K+-ATPase was examined by immunocytochemistry. To elucidate the effect of CD on cell adhesion property, same numbers of High- and Low-CD cells (150 cells/mm2) were seeded on individual culture plates and cellular adhesion and proliferation were evaluated. Cell numbers were evaluated by Cell Titer-Glo® after 3 hours of seeding. In addition, the cell proliferative ability of the High- and Low-CD cells was evaluated by Click-iT® EdU cell proliferation assays after 6 and 9 hours.
Both High- and Low-CD cells showed regular CEC morphology (regular polygonal monolayer) and expressed ZO-1 and Na+/K+-ATPase on their plasma membranes. The number of attached High-CD cells was significantly higher compared to that of Low-CD cells after 3 hours (140.8±0.3%) (p<0.01). A significantly large number of EdU-positive proliferating cells was detected in the High-CD cells compared to Low-CD cells after 6 and 9 hours (139.3±0.1% and 159.3±0.2%, respectively) (p<0.01).
The findings of the present study indicate that High-CD cells exhibited higher adhesion and proliferation properties compared to Low-CD cells. Our results suggest that cell density might be one of the indexes of product specification for future CEC transplantation.
This PDF is available to Subscribers Only