Abstract
Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bonanno JA (2012) Molecular mechanisms underlying the corneal endothelial pump. Exp Eye Res 95(1):2–7. https://doi.org/10.1016/j.exer.2011.06.004
Eghrari AO, Riazuddin SA, Gottsch JD (2015) Overview of the cornea: Structure, function, and development. Prog Mol Biol Transl Sci 134:7–23. https://doi.org/10.1016/bs.pmbts.2015.04.001
DelMonte DW, Kim T (2011) Anatomy and physiology of the cornea. J Cataract Refract Surg 37(3):588–598. https://doi.org/10.1016/j.jcrs.2010.12.037
Schneider AI, Maier-Reif K, Graeve T (1999) Constructing an in vitro cornea from cultures of the three specific corneal cell types. In Vitro Cell Dev Biol Anim 35(9):515–526. https://doi.org/10.1007/s11626-999-0062-0
Zieske JD, Mason VS, Wasson ME, Meunier SF, Nolte CJ, Fukai N, Olsen BR, Parenteau NL (1994) Basement membrane assembly and differentiation of cultured corneal cells: Importance of culture environment and endothelial cell interaction. Exp Cell Res 214(2):621–633. https://doi.org/10.1006/excr.1994.1300
Parnigotto PP, Bassani V, Montesi F, Conconi MT (1998) Bovine corneal stroma and epithelium reconstructed in vitro: Characterisation and response to surfactants. Eye (Lond) 12(Pt 2):304–310. https://doi.org/10.1038/eye.1998.70
Griffith M (1999) Functional human corneal equivalents constructed from cell lines. Science 286(5447):2169–2172. https://doi.org/10.1126/science.286.5447.2169
Griffith M (2002) Artificial human cornea. Cornea 21(2):54–61. https://doi.org/10.1097/01.ico.0000263120.68768.f8
Geesin JC, Darr D, Kaufman R, Murad S, Pinnell SR (1988) Ascorbic acid specifically increases type i and type iii procollagen messenger rna levels in human skin fibroblasts. J Invest Dermatol 90(4):420–424. https://doi.org/10.1111/1523-1747.ep12460849
Chan D, Lamande SR, Cole WG, Bateman JF (1990) Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro. Biochem J 269:175–181
Michel M, L’Heureux N, Pouliot R, Xu W, Auger FA, Germain L (1999) Characterization of a new tissue-engineered human skin equivalent with hair. In Vitro Cell Dev Biol Anim 35(6):318–326. https://doi.org/10.1007/s11626-999-0081-x
Couture C, Zaniolo K, Carrier P, Lake J, Patenaude J, Germain L, Guérin SL (2016) The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing. Biomaterials 78:86–101. https://doi.org/10.1016/j.biomaterials.2015.11.006
Carrier P, Deschambeault A, Talbot MV, Giasson CJ, Auger FA, Guérin SL, Germain L (2008) Characterization of wound reepithelialization using a new human tissue-engineered corneal wound healing model. Invest Ophthalmol Vis Sci 49(4):1376–1385. https://doi.org/10.1167/iovs.07-0904
Proulx S, d’Arc Uwamaliya J, Carrier P, Deschambeault A, Audet C, Giasson CJ, Guérin SL, Auger FA, Germain L (2010) Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types. Mol Vis 16:2192–2201
Acknowledgements
The authors would like to thank current and former members of the LOEX and CUO-Recherche laboratories who contributed to develop and improve the foregoing protocols. This work was supported by the Canadian Institutes for Health Research (CIHR) grant MOP-12087 and FDN-143213 (L.G.), the Fondation des Pompiers du Québec pour les Grands Brûlés (FPQGB), the Fonds de Recherche du Québec-Santé (FRQS), and the Réseau de thérapie cellulaire, tissulaire et génique du Québec -ThéCell (a thematic network supported by the FRQS). The Banque d’yeux Nationale is partly supported by the Réseau de Recherche en Santé de la Vision from the FRQS. P.D. and C.C. were supported by studentships from the FRQS. L.G. is the recipient of a Tier 1 Canadian Research Chair on Stem Cells and Tissue Engineering and a Research Chair on Tissue-Engineered Organs and Translational Medicine of the Fondation de l’Université Laval.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Le-Bel, G., Desjardins, P., Couture, C., Germain, L., Guérin, S.L. (2020). The Self-assembly Approach as a Tool for the Tissue Engineering of a Bi-lamellar Human Cornea. In: Ahearne, M. (eds) Corneal Regeneration. Methods in Molecular Biology, vol 2145. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0599-8_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0599-8_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0598-1
Online ISBN: 978-1-0716-0599-8
eBook Packages: Springer Protocols