Constructing an in vitro cornea from cultures of the three specific corneal cell types

SummaryThis 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.

[1]  L. Hyldahl.  Primary cell cultures from human embryonic corneas. , 1984, Journal of cell science.

[2]  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.

[3]  P. Friedl,et al.  Isolation and long-term cultivation of human corneal endothelial cells. , 1988, Investigative ophthalmology & visual science.

[4]  B. Bernard,et al.  Human epidermis reconstructed by culture: is it "normal"? , 1986, The Journal of investigative dermatology.

[5]  M. Jumblatt.  Corneal Epithelial and Endothelial Cell Culture , 1993 .

[6]  J. Mannagh,et al.  Human corneal endothelium: growth in tissue cultures. , 1965, Archives of ophthalmology.

[7]  T. Hirone,et al.  Basal lamina formation by epidermal cells in cell culture. , 1981, Current problems in dermatology.

[8]  R. Price,et al.  Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface. , 1991, Experimental cell research.

[9]  R. Burgeson,et al.  Cellular origin of the dermal‐epidermal basement membrane , 1993, Developmental dynamics : an official publication of the American Association of Anatomists.

[10]  B. Yue,et al.  Mass culture of human corneal endothelial cells. , 1979, Archives of ophthalmology.

[11]  D. Conrad,et al.  Fetal bovine serum alternatives , 1988, Nature.

[12]  B. Coulomb,et al.  The collagen lattice: a model for studying the physiology, biosynthetic function and pharmacology of the skin , 1984, The British journal of dermatology.

[13]  A. I. Schneider,et al.  The use of an in vitro cornea for predicting ocular toxicity , 1997 .

[14]  S. Danilov,et al.  Human corneal endothelial cells: isolation, characterization and long-term cultivation. , 1988, Experimental eye research.

[15]  H. Sugihara,et al.  Reconstruction of cornea in three-dimensional collagen gel matrix culture. , 1993, Investigative ophthalmology & visual science.

[16]  F. Grinnell,et al.  Reorganization of hydrated collagen lattices by human skin fibroblasts. , 1984, Journal of cell science.

[17]  J. Scott.  Proteoglycan-fibrillar collagen interactions. , 1988, The Biochemical journal.

[18]  J. Zieske,et al.  Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. , 1994, Experimental cell research.

[19]  S. Ledbetter,et al.  Bovine corneal endothelium in vitro. Elaboration and organization and of a basement membrane. , 1982, Experimental cell research.

[20]  J. D. Cameron,et al.  Rabbit corneal epithelial cells grown in vitro without serum. , 1976, Investigative ophthalmology.

[21]  E Bell,et al.  Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[22]  N. Parenteau,et al.  Skin in Complex Culture: The Transition from “Culture” Phenotype to Organotypic Phenotype , 1993 .

[23]  R A Thoft,et al.  Basement membrane synthesis by human corneal epithelial cells in vitro. , 1994, Investigative ophthalmology & visual science.

[24]  R. Haschke,et al.  Isolation and culture of corneal cells and their interactions with dissociated trigeminal neurons. , 1982, Experimental eye research.

[25]  P. Binder,et al.  The growth of endothelium from human corneal rims in tissue culture. , 1984, Investigative ophthalmology & visual science.

[26]  D. Woodley,et al.  Production of fibronectin by epithelium in a skin equivalent. , 1987, The Journal of investigative dermatology.