Summary
This paper presents a reliable method for establishing pure cultures of the three types of corneal cells. This is believed to be the first time, corneal cells have been cultured from fetal pig corneas. Cell growth studies were performed in different media. Subcultures of the three corneal cell types were passaged until the 30th generation without their showing signs of senescence. For engineering an in vitro cornea, corneal epithelial cells were cultured over corneal stromal cells in an artificial biomatrix of collagen with an underlying layer of corneal endothelial cells. The morphology, histology, and differentiation of the in vitro cornea were investigated to determine the degree of comparability to the cornea in vivo. The in vitro construct displayed signs of transition to an organotypic phenotype of which the most prominent was the formation of two basement membranes.
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Allman, M. I.; Harper, R. A.; Yanoff, M., et al. Rabbit corneal epithelial cells grown in vitro without serum. Investig. Ophthalmol. 15:666–668; 1976.
Asselineau, D.; Bernard, B. A.; Bailly, C., et al. Human epidermis reconstructed by culture: is it “normal”? Invest. Dermatol. 86:181–186; 1986.
Baum, J. L.; Niedra, R.; Davis, C.; Yue, B. J. T. Mass culture of human corneal endothelial cells. Arch. Ophthalmol. 97:1136–1140; 1976.
Bell, E.; Iversson, B.; Merrill, C. Production of tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76:1272–1278; 1979.
Bilbo, P. R.; Nolte, C. J. M.; Oleson, M. A., et al. Skin in complex culture: the transition from “culture” phenotype to organotypic phenotype. J. Toxicol. Cutaneous Ocul. Toxicol. 12:183–196; 1993.
Chamson, A.; Germain, N.; Claudy, A., et al. Study of basement membrane formation in dermal-epidermal recombinants in vitro. Arch. Dermatol. Res. 281:267–272; 1989.
Chan, K. Y.; Haschke, R. H. Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons. Exp. Eye Res. 35:137–156; 1982.
Coulomb, B.; Dubertet, I.; Merrill, C., et al. The collagen lattice: a model for studying the physiology, biosynthetic function and pharmacology of the skin. Br. J. Dermatol. 111:83–87; 1994.
Engelmann, K. J.; Böhnke, M.; Friedl, P. Isolation and long-term cultivation of human corneal endothelial cells. Investig. Ophthalmol. Vis. Sci. 29:1656–1662; 1988.
Forbes, D. J.; Pozos, R. S.; Nelson, J. D.; Lima, P. H. Characterization of rat corneal epithelium maintained in tissue culture. Curr. Eye Res. 3:1471–1479; 1984.
Fusenig, N. E.; Breitkreutz, D.; Dzarlieva, R. T., et al. Epidermal cell differentiation and malignant transformation in culture. Cancer Forum 6:209–240; 1982.
Fusenig, N. E., ed.. Cell interaction and epithelial differentiation. In: Culture of epithelial cells. New York: Wiley-Liss; 1992:25–57.
Glassman, A. B.; Coles, W. H.; Bennett, C. E. Corneal endothelium: A modified method for cultivation. In Vitro 15:873–876; 1979.
Grinnell, F.; Lamke, C. Reorganization of hydrated collagen lattices by human skin fibroblasts. Cell Sci. 66:51–63; 1984.
Grinnell, F.; Takashima, A.; Lamke Seymour, C. L. Morphological appearance of epidermal cells cultured on fibroblast-reorganized collagen gels. Cell Tissue Res. 246:13–21; 1986.
Hirone, T.; Taniguchi, S. Basal lamina formation by epidermal cells in cell culture. Curr. Probl. Dermatol. 10:159–169; 1980.
Hyldahl, L. Primary cell cultures from human embryonic corneas. Cell Sci. 66:343–351; 1984.
Jayme, D. W.; Epstein, D. A.; Conrad, D. R. Fetal bovine serum alternatives. Nature (Lond) 334:547–548; 1988.
Jumblatt, M. M. Corneal epithelial and endothelial cell culture. In: Goldberg, A. M., ed. Alternative methods in toxicology. Volume 1A. New York: Academic Press Inc.; 1993:94–99.
MacCallum, D. K.; Lillie, J. H.; Scaletta, K. H., et al. Bovine corneal endothelium in vitro. Exp. Cell Res. 139:1–13; 1982.
Mannagh, J.; Irving, A. R. Human corneal endothelial growth in tissue cultures. Arch. Ophthalmol. 74:844–851; 1965.
Marinkovich, M. P.; Keene, D. R.; Rimberg, C. S.; Burgeson R. E. Cellular origin of the dermal-epidermal basement membrane. Dev. Dyn. 197:255–267; 1993.
Minami, Y.; Sugihara, H.; Oono, S. H. Reconstruction of cornea in three-dimensional collagen gel matrix culture. Investig. Ophthalmol. Vis. Sci. 34:2316–2324; 1993.
Miltenburger, H. G. Zur Qualitätssicherung in tierischen Zellkultursystemen. In: Tierische Zellkulturen, 2. BMFT-Statusseminar. Bundesministerium für Forschung und Technologie; 1985:29–57.
Nayak, S. K.; Binder, P. S. The growth of endothelium from human corneal rims in tissue culture. Investig. Ophthalmol. Vis. Sci. 25:1213–1216; 1984.
Ohji, M.; SundarRaj, N.; Hassell, J. R.; Thoft, A. Basement membrane synthesis by human corneal epithelial cells in vitro. Investig. Ophthalmol. Vis. Sci. 25:479–485; 1994.
O’Keefe, E. J.; Woodley, D. T.; Falk, R. J., et al. Production of fibronectin by epithelium in a skin equivalent. Investig. Dermatol. 88:634–639; 1987.
Pistov, M. Yu.; Sandovnikova, E. Yu.; Danilov, S. M. Human corneal endothelial cells: isolation, characterization and long-term cultivation. Exp. Eye Res. 47:403–414; 1988.
Pond, W. G.; Houpt, K. A., ed. The biology of the pig. Ithaca and London, Cornell University Press, 1. Auflage; 1978.
Romeis, B., ed. Mikroskopische technik. München-Wien-Baltimore: Urban & Schwarzenberg; 1989.
Schafer, I. A.; Kovach, M.; Price, R. L.; Fratianne, R. B. Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen and heparan sulfate proteoglycan at the basal cell surface. Exp. Cell Res. 195:443–457; 1991.
Schermer, A.; Galvin, S.; Sun, T. T. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. Cell Biol. 103:49–62; 1986.
Schneider, A. I. Entwicklungen zu einem In-vitro-Cornea-Modell im Hinblick auf eine Alternative zum Draize-Test. Ph.D. Dissertation, University of Hohenheim; 1995.
Schneider, A. I.; Maier-Reif, K.; Graeve, T. The use of an in-vitro cornea for predicting ocular toxicity. In Vitro Toxicol. 10/3:309–318; 1997.
Scott, J. E. Proteoglycan-fibrillar collagen interactions. Biochem. J. 252:313–323; 1988.
Zieske, J. D.; Mason, V. S.; Wasson, M. E., et al. Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp. Cell Res. 214:621–632; 1994.
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Schneider, A.I., Maier-Reif, K. & Graeve, T. Constructing an in vitro cornea from cultures of the three specific corneal cell types. In Vitro Cell.Dev.Biol.-Animal 35, 515–526 (1999). https://doi.org/10.1007/s11626-999-0062-0
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DOI: https://doi.org/10.1007/s11626-999-0062-0