Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.

Arisa Mitani,Naoko Takahira,Saori Nakao,Sachi Matsushita,Yasuhito Hayashi,Natsuki Matsushita,Takeshi Kobayashi,Yuichi Ohashi,Atsushi Shiraishi,Alexander V Ljubimov

doi:10.1371/journal.pone.0222454

Arisa Mitani, Naoko Takahira + Show 8 more

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https://doi.org/10.1371/journal.pone.0222454

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Abstract

PurposeTo present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line.MethodsiHCjECs were developed to induce Simian Virus 40 large T-antigen (SV40LT) by incorporating lentivirus in a tetracycline (Tet)-regulated gene-expression system into primary cultures of human conjunctival epithelial cells. The population doubling time and morphology of the iHCjECs were analyzed. The expressions of CK13, CK19, CK12, and MUC1, MUC4, MUC16, and MUC5AC were determined by real time PCR and immunohistochemically under different culture conditions. The organotypic culture model in which iHCjECs were cultured on rabbit conjunctival fibroblast-embedded collagen gel was used to characterize the iHCjECs.ResultsThe iHCjECs cultured with doxycycline (Dox) continued to proliferate for at least 20 passages and had a cobblestone-like appearance. The expressions of CK13 and CK19 but not CK12 were detected in the iHCjECs, and the expression of CK13 increased in culture media lacking Dox (Dox-). The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS. Stratified iHCjECs were observed in organotypic culture at 5 days.ConclusionThe iHCjECs had high proliferation rates and abilities to control the differentiation potency to control the expression of SV40 LT-antigen with Tet-regulated gene-expression system. They are able to express the mucin gene repertoire of their native epithelia. The iHCjECs can be a useful experimental cell line to study conjunctival epithelial cell characteristics and for pathophysiological and toxicological studies.

Highlights

The surface of the eye is exposed to the outside world and is subject to infections, drying, and injury
The expressions of CK13 and CK19 but not CK12 were detected in the immortalized human conjunctival epithelial cell (iHCjEC), and the expression of CK13 increased in culture media lacking Dox (Dox-)
The expressions of MUC1, MUC4, MUC16, and MUC5AC were detected in iHCjECs, and a relatively strong immunostaining of MUC5AC was detected with Dox(-) added 5% FBS

Summary

Introduction

The surface of the eye is exposed to the outside world and is subject to infections, drying, and injury. The conjunctival epithelial cells on the surface protect the eye by maintaining a healthy ocular surface. There are several ways to obtain human conjunctival epithelial cells for investigations on the physiology of the surface of the eye. Primary cultures of human conjunctival epithelial cells have been shown to have the ability to produce and secrete mucin-type glycoproteins [3,4,5]. These primary cultures are prepared from human conjunctiva biopsy specimens, the tissue availability is limited and the amount and longevity of the cells are limited

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