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Marwan Ali, Dhara Shah, Zeeshan Pasha, Vinay Kumar Aakalu; Immortalization of Human Lacrimal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):686.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Study of human lacrimal epithelial cell biology is limited by poor access to tissue and difficulty with maintaining primary cultures. A better understanding of lacrimal epithelial biology would advance efforts to develop treatments for dry eye disease and other lacrimal gland diseases. In this study, we utilized viral vectors expressing hTERT to develop an immortalized human lacrimal epithelial cell line.
Methods: Primary human Lacrimal Epithelial (hLE) cells from passage 3 to 5 were immortalized using Lentiviral vectors expressing GFP-tagged hTERT gene. Cultured primary hLE cells were transfected with pLenti-GIII-CMV-hTERT-GFP-2A-Purovirus at Multiplicity Of Infection (MOI) of 1, 1.5 and 2 in the presence of 0.8ug/ml Polybrene. The next day, the viral supernatant was removed and replaced with fresh complete HepatoStim growth medium. After three days, 10ug/ml Puromycin Dihydrochloride was added to medium for selection of immortalized hLE cells. Immortalized hLE cells were then viewed using bright field and fluorescent microscopies to detect GFP signal of transfected hLE cells. Successful transfection of different hLE cells from the different passages was analyzed using RT-PCR and fluorescence microscopy. GFP labeled cells were selected using FACS and subcultured. Isolated cells were passaged and followed for a period of 100 days. RT-PCR and immunofluorescence analysis were conducted to evaluate lacrimal epithelial phenotype and telomerase activity for 20 passages.
Results: Successful immortalization of human lacrimal epithelial cells using a lentiviral hTERT vector with GFP reporting was demonstrated through maintenance of epithelial cells through multiple passages, evidence of integration of the viral vector, stable expression of GFP and maintenance of lacrimal epithelial gene expression patterns and immunophenotyping.
Conclusion: We have successfully developed an immortalized human lacrimal epithelial cell line which can be used for future study in developing cell based therapies for dry eye disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Immortalized Human Lacrimal Epithelial Cells Passage5 (MOI 1)
Immortalized Human Lacrimal Epithelial Cells Passage3 (MOI 1.6)
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