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K. Nishitsuka, Y. Kashiwagi, C. Kanno, T. Yamamoto, H. Takamura, H. Yamashita; Immortalization of Several Human Eye-Derived Cells Using Human Papilloma Virus 16 E6 and E7. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2988.
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© ARVO (1962-2015); The Authors (2016-present)
Eyes consist of several tissues, and to investigate tissue function and eye diseases, cell culture systems are very useful. In this study, we sought to establish cell lines or cell strains derived from human tissues.
This study was approved by the Yamagata University Faculty of Medicine. We cultured primary cells derived from human tissues removed as surgical specimens after securing written permission from the subjects and then transfected immortalized genes (human papilloma virus 16 E6 and E7 genes) into the primary cells using a recombinant retrovirus system. We then selected transfected cells using G418 solution. To compare the biological features of primary and immortalized cells, we observed the morphological features and the mRNA expression patterns of collagen and CD antigen genes.
We obtained primary cells from human tissue, including lens epithelium, cornea stroma, sclera stroma, choroid, ciliary body, iris, trabecular meshwork, vitreous, and conjunctiva, and transfected immortalized genes. We obtained 9 clones of the corneal stroma-derived cell strains (HCs10), 10 clones of the sclera-derived cell strains (HSc10), and 11 clones of vitreous-derived cell strains (HV64 and HV65). These cell strains could be passaged up to 20 times. The HCs10, HSc10, HV64, and HV65 cell-doubling times were 65, 60, 58, and 76 h, respectively. Both the corneal stroma- and sclera-derived cells expressed transcripts of collagen types I to VIII. The vitreous-derived cells expressed collagen types I and II and CD11b, 14, 68, 204, and 206 transcripts. The cellular forms and mRNA expression patterns of collagen and CD antigens were identical between the primary cells and cell strains.
We succeeded in deriving human cell strains from several eye tissues (cornea stroma, sclera stroma, and vitreous) using HPV16 E6/E7 immortalized genes. These primary cells and cell strains can be used in experiments testing collagen and CD mRNA expression, as well as some of the cellular characteristics.
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