Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets

Lily Wei Chen; Yan-Ming Chen; Chia-Ju Lu; Mei-Yun Chen; Szu-Yuan Lin; Fung-Rong Hu; Wei-Li Chen

doi:10.4103/tjo.tjo_101_17

Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets

Taiwan J Ophthalmol. 2017 Oct-Dec;7(4):205-212. doi: 10.4103/tjo.tjo_101_17.

Authors

Lily Wei Chen¹, Yan-Ming Chen^{1

2}, Chia-Ju Lu¹, Mei-Yun Chen¹, Szu-Yuan Lin³, Fung-Rong Hu^{1

4}, Wei-Li Chen^{1

4}

Affiliations

¹ Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.
² Department of Ophthalmology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan.
³ Department of Ophthalmology, Cathay General Hospital, Taipei, Taiwan.
⁴ Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan.

Abstract

Purpose: The aim of this study is to evaluate the effects of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of rabbit limbal stem cells cultivated in vitro, and to find out the proper timing of air-lifting before transplantation as limbal epithelial cell sheets for the treatment of limbal insufficiency.

Materials and methods: Limbal epithelial cells were isolated from the limbus of New Zealand white rabbits and cultivated in vitro. After the cells became confluent, different durations of air-lifting (0, 1, 2, 4, and 7 days) were performed. At the end of cultivation, immunohistochemistry on cryosections was performed and observed by fluorescein microscopy and in vitro confocal microscopy for cytokeratins (K3, K10, K12, K13, and K14), junctional and structural proteins (ZO-1, p120, and actin) and stem cell markers (ABCG2 and P63). Scanning electron microscopy was used for observing the microstructure of superficial cells. Transepithelial electrical resistance (TEER) was used to measure the transepithelial permeability.

Results: The expression of K3, K10, K12, K13, K14, and ABCG2 showed no differences in pattern and location among different groups of airlifting. A time-dependent increase in corneal epithelial thickness was found after air-lifting. In vitro confocal microscopy demonstrated that K3, p120, and ZO-1 were expressed on the apical cell layer, whereas P63 and ABCG2 were expressed more on the basal epithelial layer. Scanning electron microscopy of the superficial layer demonstrated that airlifting induced time-dependent increase in the size of surface epithelial cells and triggered cellular differentiation. TEER results demonstrated a time-dependent increase of transepithelial electric resistance.

Conclusions: During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the transepithelial barrier. However, the expression of cellular junctional, stem cell and cytokeratin markers seems to have no obvious differences in pattern and localization.

Keywords: Airlifting; cultivated cell sheets; cytokeratin; limbus; stem cell.