RRC ID 57648
Author Mitani A, Kobayashi T, Hayashi Y, Matsushita N, Matsushita S, Nakao S, Takahira N, Shiraishi A, Ohashi Y.
Title Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line.
Journal PLoS One
Abstract PURPOSE:To present the properties of a newly developed immortalized human conjunctival epithelial cell (iHCjEC) line.
METHODS:iHCjECs 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.
RESULTS:The 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.
CONCLUSION:The 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.
Volume 14(9)
Pages e0222454
Published 2019-9-11
DOI 10.1371/journal.pone.0222454
PII PONE-D-19-10471
PMID 31509592
PMC PMC6738650
MeSH Antigens, Viral, Tumor / metabolism* Cell Culture Techniques / methods* Cell Line / metabolism Cell Line, Transformed / metabolism Cells, Cultured Conjunctiva / metabolism Doxycycline / metabolism Doxycycline / pharmacology Epithelial Cells / metabolism Gene Expression / genetics Gene Expression Regulation / genetics Humans RNA, Messenger / genetics Simian virus 40 / metabolism*
IF 2.74
Times Cited 0
DNA material CS-CA-MCS (RDB05963) pCMV-VSV-G-RSV-Rev (RDB04393) pCAG-HIVgp (RDB04394)
Human and Animal Cells 293T(RCB2202)