Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies.

PURPOSE To develop an in vitro model of human corneal epithelium that can be propagated in serum-free medium that is tissue specific, species specific, and continuously available. METHODS Primary explant cultures from human cadaver donor corneas were generated and subsequently infected with Adeno 12-SV40 (Ad12-SV40) hybrid virus or transfected with plasmid RSV-T. RESULTS Several lines of human corneal epithelial cells with extended life span were developed and characterized. Propagation of both primary cultures and lines with extended life span, upon collagen membranes at an air-liquid interface, promoted multilayering, more closely approximating the morphology observed in situ. CONCLUSIONS In vitro models, using primary cultures of corneal epithelium and lines of corneal epithelial cells with extended life span, retain a variety of phenotypic characteristics and may be used as an adjunct to ocular toxicology studies and as a tool to investigate corneal epithelial cell biology.

[1]  J. Northrop,et al.  Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P M Steinert,et al.  Molecular and cellular biology of intermediate filaments. , 1988, Annual review of biochemistry.

[3]  J. Rhim,et al.  Malignant human papillomavirus type 16-transformed human keratinocytes exhibit altered expression of extracellular matrix glycoproteins. , 1991, Cancer research.

[4]  John H. Draize,et al.  METHODS FOR THE STUDY OF IRRITATION AND TOXICITY OF SUBSTANCES APPLIED TOPICALLY TO THE SKIN AND MUCOUS MEMBRANES , 1944 .

[5]  D. Pim,et al.  Monoclonal antibodies specific for simian virus 40 tumor antigens , 1981, Journal of virology.

[6]  D. Doughman,et al.  Prolonged Donor Cornea Preservation In Organ Culture: Long‐Term Clinical Evaluation , 1982, Transactions of the American Ophthalmological Society.

[7]  M. Lechner,et al.  Human epithelial cells immortalized by SV40 retain differentiation capabilities in an in vitro raft system and maintain viral DNA extrachromosomally. , 1991, Virology.

[8]  Doughman Dj.  Prolonged donor cornea preservation in organ culture: long-term clinical evaluation. , 1980 .

[9]  A. Schermer,et al.  Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells , 1986, The Journal of cell biology.

[10]  J. Rhim,et al.  Neoplastic transformation of chimpanzee cells induced by adenovirus type 12--simian virus 40 hybrid virus. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[11]  T. Sun,et al.  Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states , 1977, Nature.

[12]  J. Friend,et al.  Comparison of limbal and peripheral human corneal epithelium in tissue culture. , 1988, Investigative ophthalmology & visual science.

[13]  K K Sanford,et al.  Neoplastic transformation of human epidermal keratinocytes by AD12-SV40 and Kirsten sarcoma viruses. , 1985, Science.

[14]  C. Harris,et al.  Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells. , 1991, Cancer research.

[15]  L A Rogers,et al.  The American Type Culture Collection , 1925, Nature.

[16]  Dennis Swanston,et al.  Book Review: A Critical Evaluation of Alternatives to Acute Ocular Irritation Testing , 1987 .

[17]  John M. Frazier,et al.  In vitro toxicity testing : applications to safety evaluation , 1992 .

[18]  S. Hainsworth.  Modified culture method for human corneal epithelial cells , 1991 .

[19]  P S Binder,et al.  Air/liquid corneal organ culture: a light microscopic study. , 1991, Current eye research.

[20]  O. Flint,et al.  In Vitro Test Validation: A House Built on Sand , 1992 .

[21]  文秀 江波戸.  Comparison of central and peripheral human corneal epithelium in tissue culture , 1988 .

[22]  E. Fuchs,et al.  A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases , 1991, The Journal of cell biology.