Reconstructed Human Cornea Produced in vitro by Tissue EngineeringGermain L.a · Auger F.A.a · Grandbois E.a · Guignard R.a · Giasson M.b · Boisjoly H.c · Guérin S.L.b
aLaboratoire d’Organogénèse Expérimentale (LOEX), Hôpital du Saint-Sacrement, Québec et Département de Chirurgie, Université Laval, Sainte-Foy, bLaboratoire d’Endocrinologie Moléculaire, Centre de Recherche du CHUL, Québec, et cDépartement d’Ophtalmologie, Hôpital Maisonneuve-Rosemont, Université de Montréal, Montréal, Canada
Do you have an account?
- Rent for 48h to view
- Buy Cloud Access for unlimited viewing via different devices
- Synchronizing in the ReadCube Cloud
- Printing and saving restrictions apply
Rental: USD 8.50
Cloud: USD 20.00
The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson’s trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as β1, α3, α4, α5, and α6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4–5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the α4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.
Hogan MJ, Zimmerman LE: The cornea and sclera; in Hogan MJ, Zimmerman LE (eds): Ophthamlic Pathology. An Atlas and Textbook. Philadelphia, Saunders, 1962, pp 277–290.
Scroggs MW, Klintworth GK: Normal eye and ocular adnexa; in Sternberg SS (ed): Histology for Pathologists. New York, Raven Press, 1992, pp 903–911.
- Kruse FE, Tseng SCG: A tumor promoter-resistant subpopulation of progenitor cells is larger in limbal epithelium than in corneal epithelium. Invest Ophthalmol Vis Sci 1993;34:2501–2511.
- Lavker RM, Dong G, Cheng SZ, Kudoh K, Cotsarelis G, Sun TT: Relative proliferative rates of limbal and corneal epithelia. Invest Ophthalmol Vis Sci 1991;32:1864–1875.
- Zieske JD, Bukusoglu G, Yankauckas MA: Characterization of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci 1992;33:143–152.
- Huang AJW, Tseng SCG: Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32:96–105.
- Lindberg K, Brown ME, Chaves HV, Kenuon KR, Rhienwald JG: In vitro propagation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci 1993;34:2672–2679.
- Ebato B, Friend J, Thoft RA: Comparison of limbal and peripheral human corneal epithelium in tissue culture. Invest Ophthalmol Vis Sci 1988;29:1533–1537.
- Zieske JD, Mason VS, Wasson ME, Meunier SF, Nolte CJM, Fukai N, Olsen BR, Parenteau NL: Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp Cell Res 1994;214:621–633.
- Ljubimov AV, Burgeson RE, Butkowski RJ, Michael AF, Sun T, Kenney MC: Human corneal basement membrane heterogeneity: Topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 1995;72:461–473.
- Gailit JO, Clark RAF: Integrins. Adv Dermatol 1993;8:129–153.
- Lauweryns B, van den Oord JJ, Valpes R, Foets B, Missotten L: Distribution of very late activation integrins in the human cornea. An immunohistochemical study using monoclonal antibodies. Invest Ophthalmol Vis Sci 1991;32:2079–2085.
- Tervo K, Tervo T, van Setten G, Virtanen I: Integrins in human corneal epithelium. Cornea 1991;10:461–465.
- Stepp MA, Spurr-Michaud S, Gipson IK: Integrins in the wounded and unwounded stratified squamous epithelium of the cornea. Invest Ophthalmol Vis Sci 1993;34:1829–1844.
- Masur SK, Cheung JKH, Antohi S: Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes. Invest Ophthalmol Vis Sci 1993;34:2690–2698.
- Maldonada BA, Furcht LT: Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. Invest Ophthalmol Vis Sci 1995;36:2120–2126.
- Latvala T, Paallysaho T, Tervo K, Tervo T: Distribution of α6 and β4 integrins following epithelial abrasion in the rabbit cornea. Acta Ophthalmol Scand 1996;74:21–25.
- Stepp MA, Zhu L: Upregulation of α9 integrin and tenascin during epithelial regeneration after debridement in the cornea. J Histochem Cytochem 1997;45:189–201.
- Stepp MA, Zhu L, Cranfill R: Changes in β4 integrin expression and localization in vivo in response to corneal epithelial injury. Invest Ophthalmol Vis Sci 1996;37:1593–1601.
- Stepp MA, Spurr-Michaud S, Tisdale A, Elwell J, Gipson IK: Alpha6β4 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci USA 1990;87:8970–8974.
Minami Y, Sugihara H, Oono S: Reconstruction of cornea in three-dimensional collagen gel matrix culture. Invest Ophthalmol Vis Sci 1993;34:2315–2324.
- Tsai RJ, Ho Y, Chen J: The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent. Invest Ophthalmol Vis Sci 1994;35:2865–2875.
Gipson IK, Grill SM: A technique for obtaining sheets of intact rabbit corneal epithelium. Invest Ophthalmol Vis Sci 1982;23:269–273.
Germain L, Rouabhia M, Guignard R, Carrier L, Bouvard V, Auger FA: Improvement of human keratinocyte isolation and culture using thermolysin. Burns 1993;2:99–104.
- Green H, Kehinde O, Thomas J: Growth of cultured human epidermal cells into a multiple epithelial suitable for grafting. Proc Natl Acad Sci USA 1979;76:5665–5668.
Gallop PM, Seifter S: Preparation and properties of soluble collagens. Methods Enzymol 1963;6:635–641.
Auger FA, López-Valle CA, Guignard R, Tremblay N, Noël B, Goulet F, Germain L: Skin equivalent produced with human collagen. In Vitro Cell Dev Biol-Animal 1995;31:432–439.
Germain L, auger F: Tissue engineered biomaterials: Biological and mechanical characteristics. Encyclopedic Handbook of Biomaterials and Bioengineering, pt B: Applications. New York, Marcel Dekker, 1995, vol 1, pp 699–734.
- Michel M, L’Heureux N, Auger FA, Germain L: From newborn to adult: Phenotypic and functional properties of skin equivalent and human skin as a function of donor age. J Cell Physiol 1997;171:179–189.
- L’Heureux N, Pâquet S, Labbé R, Germain L, Auger FA: A completely biological tissue-engineered human blood vessel. FASEB J 1998;12:47–56.
Paallysaho T, Tervo T, Virtanen I, Tervo K: Integrins in the normal and healing epithelium. Acta Ophthalmol Suppl 1992;202:22–25.
- Sheppard D: Epithelial integrins. Bioessays 1996;18:655–660.
Auger FA, Rouabhia M, Goulet F, Berthod F, Moulin V, Germain L: Tissue engineered human skin substitutes developed from collagen populated hydrated gels: Clinical and fundamental applications. Med Biol Eng Comp 1988;36(6):801–802.
Gay R, Swiderek M, nelson D: The living dermal equivalent as an in vitro model for predicting ocular irritation. J Toxicol Cut Ocular Toxicol 1992;11:47–68.
Article / Publication Details
Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.