Cloning and characterization of cell strains derived from human corneal stroma and sclera

• Published in: Japanese journal of ophthalmology 2010-01, Vol.54 (1), p.74-80
• Main Authors: Kashiwagi, Yoshiko, Nishitsuka, Koichi, Namba, Hiroyuki, Kamiryo, Masaru, Takamura, Hiroshi, Yamashita, Hidetoshi
• Format: Article
• Language: English
• Subjects: Aged; Cells, Cultured; Cloning, Molecular; Collagen - genetics; Corneal Stroma - cytology; Corneal Stroma - metabolism; Female; Fibroblasts - cytology; Fibroblasts - metabolism; Gene Expression Regulation, Viral - physiology; Glucuronosyltransferase - genetics; Humans; Hyaluronan Synthases; Hyaluronic Acid - metabolism; Laboratory Investigation; Matrix Metalloproteinases - genetics; Medicine; Medicine & Public Health; Oncogene Proteins, Viral - genetics; Ophthalmology; Papillomavirus E7 Proteins - genetics; Repressor Proteins - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Sclera - cytology; Sclera - metabolism; Tissue Inhibitor of Metalloproteinases - genetics; Transfection
• ISSN: 0021-5155; 1613-2246
• DOI: 10.1007/s10384-009-0749-5

Purpose To establish human corneal stroma- and sclera-derived cells as models for studying diseases of the anterior segment of the eye. Methods Using a recombinant retrovirus system, we transfected human papilloma virus 16 E6 and E7 (HPV16 E6/E7) into human corneal stroma- and sclera-derived cells. The primary cells and established cell strains were characterized by assessing the mRNA expression of collagen, matrix metalloproteinase, and tissue inhibitor of metalloproteinase by reverse transcription-polymerase chain reaction. We also examined the effects of inflammatory cytokines on hyaluronan synthase expression and hyaluronan products. Results Both a corneal stroma-derived cell strain, Cs3, and a sclera-derived cell strain, Sc1, were obtained, and both cell strains could be passaged up to 25 times. The mRNA expression pattern observed in the primary cells was identical to that observed in the cell strains. Hyaluronan synthase 1 and 2 mRNAs were increased by transforming growth factor β and platelet-derived growth factor BB. Significant differences were observed between the hyaluronan products with and without cytokine treatment. Conclusion Cell strains derived from corneal stroma and sclera fibroblast cells can be established using HPV16 E6/E7 immortalized genes of the same origin. The phenotypic cell characteristics did not change after transfection, immortalization, or successive passages in culture.