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Primary Culture of Cornea-Limbal Epithelial Cells In Vitro

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Corneal Regeneration

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2145))

Abstract

The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.

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References

  1. Thoft RA, Friend J (1983) The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442–1443

    CAS  PubMed  Google Scholar 

  2. Koizumi N, Cooper LJ, Fullwood NJ et al (2002) An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture. Investig Ophthalmol Vis Sci 43:2114–2121

    Google Scholar 

  3. Ghoubay-Benallaoua D, Basli E, Goldschmidt P et al (2011) Human epithelial cell cultures from superficial limbal explants. Mol Vis 17:341–354

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kolli S, Lako M, Figueiredo F et al (2008) Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane. Regen Med 3:329–342. https://doi.org/10.2217/17460751.3.3.329

    Article  CAS  PubMed  Google Scholar 

  5. Liu S, Li J, Wang C et al (2006) Human limbal progenitor cell characteristics are maintained in tissue culture. Ann Acad Med Singap 35:80–86

    PubMed  Google Scholar 

  6. Pellegrini G, Traverso CE, Franzi AT et al (1997) Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349:990–993. https://doi.org/10.1016/S0140-6736(96)11188-0

    Article  CAS  PubMed  Google Scholar 

  7. Espana EM, Romano AC, Kawakita T et al (2003) Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Investig Ophthalmol Vis Sci 44:4275–4281. https://doi.org/10.1167/iovs.03-0089

    Article  Google Scholar 

  8. Spurr SJ, Gipson IK (1985) Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone. Invest Ophthalmol Vis Sci 26:818–827

    CAS  PubMed  Google Scholar 

  9. Kubo M, Sonoda Y, Muramatsu R, Usui M (2001) Immunogenicity of human amniotic membrane in experimental xenotransplantation. Invest Ophthalmol Vis Sci 42:1539–1546

    CAS  PubMed  Google Scholar 

  10. Koizumi N, Inatomi T, Sotozono C, et al (2000) Growth factors in amniotic membrane Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res. 20(3) : 173–177

    Google Scholar 

  11. Moriyama T, Asahina I, Ishii M et al (1994) Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp Cell Res 4:415–427

    Google Scholar 

  12. Fukuda K, Chikama T, Nakamura M, Nishida T (1999) Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva. Cornea 18:73–79. https://doi.org/10.1097/00003226-199901000-00013

    Article  CAS  PubMed  Google Scholar 

  13. Chen B, Jones RR, Mi S et al (2012) The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair. Soft Matter 8:8379. https://doi.org/10.1039/c2sm26175h

    Article  CAS  Google Scholar 

  14. Puck TT, Marcus PI, CieciuraI SJ (1956) Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer. J Exp Med 103:273–283. https://doi.org/10.1084/jem.103.2.273

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ehmann UK, Stevenson MA, Calderwood SK, DeVries JT (1998) Physical connections between feeder cells and recipient normal mammary epithelial cells. Exp Cell Res 243:76–86. https://doi.org/10.1006/excr.1998.4054

    Article  CAS  PubMed  Google Scholar 

  16. Burroughs J, Gupta P, Blazar BR, Verfaillie CM (1994) Diffusible factors from the murine cell line M2-10B4 support human in vitro hematopoiesis. Exp Hematol 22:1095–1101

    CAS  PubMed  Google Scholar 

  17. Tseng SC, Kruse FE, Merritt J, Li DQ (1996) Comparison between serum-free and fibroblast-cocultured single-cell clonal culture systems: evidence showing that epithelial anti-apoptotic activity is present in 3T3 fibroblast-conditioned media. Curr Eye Res 15:973–984. https://doi.org/10.3109/02713689609017643

    Article  CAS  PubMed  Google Scholar 

  18. Kim MK, Lee JL, Oh JY et al (2008) Efficient cultivation conditions for human limbal epithelial cells. J Korean Med Sci 23:864. https://doi.org/10.3346/jkms.2008.23.5.864

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pellegrini G, Golisano O, Paterna P et al (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 145:769–782. https://doi.org/10.1083/jcb.145.4.769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343. https://doi.org/10.1016/s0092-8674(75)80001-8

    Article  CAS  PubMed  Google Scholar 

  21. Sun TT, Green H (1977) Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature 269:489–493. https://doi.org/10.1038/269489a0

    Article  CAS  PubMed  Google Scholar 

  22. Yaeger PC, Stiles CD, Rollins BJ (1991) Human keratinocyte growth-promoting activity on the surface of fibroblasts. J Cell Physiol 149:110–116. https://doi.org/10.1002/jcp.1041490114

    Article  CAS  PubMed  Google Scholar 

  23. Yokoo S, Yamagami S, Yanagi Y et al (2005) Human corneal endothelial cell precursors isolated by sphere-forming assay. Invest Ophthalmol Vis Sci 46:1626–1631. https://doi.org/10.1167/iovs.04-1263

    Article  PubMed  Google Scholar 

  24. Brejchova K, Trosan P, Studeny P et al (2018) Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions. Exp Eye Res 176:20–28. https://doi.org/10.1016/j.exer.2018.06.019

    Article  CAS  PubMed  Google Scholar 

  25. Zamudio A, Wang Z, Chung S-H, Wolosin JM (2016) Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions. Exp Eye Res 145:48–57. https://doi.org/10.1016/j.exer.2015.10.021

    Article  CAS  PubMed  Google Scholar 

  26. Lekhanont K, Choubtum L, Chuck RS et al (2009) A serum- and feeder-free technique of culturing human corneal epithelial stem cells on amniotic membrane. Mol Vis 15:1294–1302

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work is supported by Science Foundation Ireland cofunded by ERDF, grant no 12/RC/2275_P2.

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Authors and Affiliations

  1. National Institute for Cellular Biotechnology and SSPC-SFI, Centre for Pharmaceuticals, Dublin City University, Dublin, Ireland

    Finbarr O’Sullivan

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  1. Finbarr O’Sullivan
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Correspondence to Finbarr O’Sullivan .

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Editors and Affiliations

  1. Trinity Centre for Biomedical Engineering, Trinity College Dublin, Dublin, Ireland

    Mark Ahearne

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O’Sullivan, F. (2020). Primary Culture of Cornea-Limbal Epithelial Cells In Vitro. 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_3

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  • DOI: https://doi.org/10.1007/978-1-0716-0599-8_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0598-1

  • Online ISBN: 978-1-0716-0599-8

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