August 1993
Volume 34, Issue 9
Free
Articles  |   August 1993
In vitro propagation of human ocular surface epithelial cells for transplantation.
Author Affiliations
  • K Lindberg
    Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.
  • M E Brown
    Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.
  • H V Chaves
    Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.
  • K R Kenyon
    Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.
  • J G Rheinwald
    Department of Research and Development, BioSurface Technology, Inc., Cambridge, Massachusetts 02139.
Investigative Ophthalmology & Visual Science August 1993, Vol.34, 2672-2679. doi:
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      K Lindberg, M E Brown, H V Chaves, K R Kenyon, J G Rheinwald; In vitro propagation of human ocular surface epithelial cells for transplantation.. Invest. Ophthalmol. Vis. Sci. 1993;34(9):2672-2679.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To examine the possibility that ocular surface epithelial cells might be grown in culture for use as grafts. METHODS: The proliferative capacity of epithelial cells cultured from the conjunctiva, limbus, and central cornea of normal human eyes was compared. Single cells disaggregated from approximately 1 mm2 biopsy specimens were serially cocultured with lethally irradiated mouse 3T3 fibroblasts. To study the cells' ability to reform a stratified epithelium, confluent limbal cultures were released as an intact cell sheet with the enzyme Dispase and transplanted to a dermal connective tissue bed in nude mice. Attachment and differentiation properties of the reconstituted epithelium were examined immunohistochemically. RESULTS: Central corneal epithelial cells could not be propagated; they senesced in first or second passage. In contrast, limbal epithelial cells exhibited a substantial (i.e., mean of 23 population doublings) and conjunctival cells a moderate (i.e., mean of 11 population doublings) proliferative capacity. Within 4 days of transplantation to the nude mouse dermis, cultured limbal epithelial cells formed an epithelium 5-6 cell layers thick. The epithelium adhered firmly to the graft bed, and deposition of the basement membrane and anchoring fibril protein collagens IV and VII and laminin was detectable immunohistochemically. The transplanted epithelium displayed limbuslike compartmental expression of keratins K3, K13, and K19, and of the enzyme enolase. CONCLUSIONS: These results support the concept that corneal epithelial stem cells are located in the limbus and indicate that cultured autologous limbal cells may function as grafts to permanently restore the corneal epithelium after severe ocular surface injury.

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