Purchase this article with an account.
Y. Ishino, Y. Sano, T. Nakamura, N. Koizumi, T. Horikiri, S. Kinoshita; Cell–Cell Contacts and Na–K Pump Sites of Cultivated Human Corneal Endothelial Cell Sheets Using Amniotic Membrane as a Carrier . Invest. Ophthalmol. Vis. Sci. 2006;47(13):73.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We reported the feasibility of cultivated human corneal endothelial cell (cHCEC) transplantation using amniotic membrane (AM) and freeze–dried AM (FDAM) as a carrier (Y Ishino et al, 2004 IOVS, 2005 ARVO). However we had not investigated cell–cell contacts and pump sites of cHCEC cultivated on these carriers yet. In this study we aimed to examine cell–cell contacts and Na–K ATPase localization in cHCEC cultivated on AM and FDAM carriers.
cHCEC (10 years old, male) from 6 passages were trypsinized, centrifugated and resuspended at a final cell seeding concentration of 6.0×103cells/mm2. Human AM deprived of amniotic epithelial cells by incubation with EDTA. For making FDAM, after incubation, resultant AM was freeze dried, vacuum packed, and sterilized with gamma–irradiation. On the resultant AM and FD–AM, the cHCEC suspension was seeded, centrifugated and cultivated for 7days at 37°C, 5%CO2. Cultures of cHCEC on AM and FD–AM were stained with alizarin red and evaluated their morphology. Immunocytochemistry evaluated cell–cell contacts by staining for ZO–1, and Na–K pump site by staining for Na–K ATPase.
The densities of cHCEC were 2433.7 (AM) and 2414.9 (FDAM) cells/mm2. CV were 39.8 (AM) and 30.1 (FDAM). Hexagonalities were 31.0 (AM) and 30.6 (FDAM) %. There were no significant difference except CV (p<0.05). ZO–1 staining demonstrated that cHCEC kept good cell–cell contacts both on AM and FDAM like HCEC in vivo. Na–K ATPase staining showed cHCEC had its expression on cell boundaries on both carriers. There is no significant ZO–1 and Na–K ATPase staining difference between cHCEC on AM and FDAM carriers.
We examined cell–cell contacts and Na–K ATPase localization in cHCEC cultivated on AM and FDAM carriers. We had shown they worked in vivo in previous studies, in the current studies, immunocytochemistry by ZO–1 and ATPase showed they had similar cell–cell contacts and Na–K ATPase localization to those in vivo.
This PDF is available to Subscribers Only