Structure and turnover rate of long-term cultured human limbal epithelial cell sheets

Abstract

 

Purpose

 

To confirm whether primary cultured human limbal epithelial cell sheets can maintain homeostasis long term with KGF and rho kinase inhibitor Y-27632.

 

Methods

 

Human limbal epithelial cells (4-8 x10⁴cells / insert) were primary cultured on plastic cell culture inserts, with a feeder layer of human mesenchymal stem cells in the bottom of a paired well. Cells were fed with supplementary hormonal epithelial medium, containing KGF and ROCK inhibitor Y-27632 but not EGF. From 5 months to 7 months, cultured medium in cell culture inserts was daily collected, and the number of cell debris was counted by hematocytometer. At 6 months, some cell sheets were treated with EdU (10 μM) for 1 day, followed by the immunostaining of cryosections and whole mount sheets with anti-EdU, antibody for immature epithelial cell marker K15, and DAPI. Nuclei in randomly selected 5 images (Obj x20, 1.48x10^-3 cm²) were counted manually by using Image J and add-in cell counter software, to estimate the total cell number in each culture insert (4.67 cm²).

 

Results

 

Each cell culture insert was estimated to contain 3.4 ± 0.1 x10⁶ cells (mean ± S.D., n=3) at 6 month, which shed 4.0 ± 0.8 x10⁴ cells every day from 5 to 7 months. K15 was expressed heterogeneously in the basal layer. EdU was mainly observed in K15 negative cells.

 

Conclusions

 

Primary human limbal epithelial cell sheets daily shed approximately 1% of total cells even after 6 months of cultivation, suggesting that these sheets maintain homeostasis.

 

 

Keywords: 482 cornea: epithelium • 721 stem cells