Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

In vitro culture and expansion of human limbal epithelial cells

Subjects

Abstract

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes 2 weeks to establish a confluent monolayer from which 3 × 106 cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Stepwise display of the procedure and culture outcomes.
Figure 2: Characterization of in vitro–expanded limbal epithelial cells on hAM.

Similar content being viewed by others

References

  1. Schermer, A., Galvin, S. & Sun, T.T. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J. Cell. Biol. 103, 49–62 (1986).

    Article  CAS  Google Scholar 

  2. Cotsarelis, G., Cheng, S.Z., Dong, G., Sun, T.T. & Lavker, R.M. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57, 201–209 (1989).

    Article  CAS  Google Scholar 

  3. Huang, A.J. & Tseng, S.C. Corneal epithelial wound healing in the absence of limbal epithelium. Invest. Ophthalmol. Vis. Sci. 32, 96–105 (1991).

    CAS  PubMed  Google Scholar 

  4. Puangsricharern, V. & Tseng, S.C. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 102, 1476–1485 (1995).

    Article  CAS  Google Scholar 

  5. Shapiro, M.S., Friend, J. & Thoft, R.A. Corneal re-epithelialization from the conjunctiva. Invest. Ophthalmol. Vis. Sci. 21, 135–142 (1981).

    CAS  PubMed  Google Scholar 

  6. Shimazaki, J., Yang, H.Y. & Tsubota, K. Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology 104, 2068–2076 (1997).

    Article  CAS  Google Scholar 

  7. Kenyon, K.R. & Tseng, S.C. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 96, 709–722 (1989).

    Article  CAS  Google Scholar 

  8. Tsai, R.J. & Tseng, S.C. Human allograft limbal transplantation for corneal surface reconstruction. Cornea 13, 389–400 (1994).

    Article  CAS  Google Scholar 

  9. Pellegrini, G., Traverso, C.E., Franzi, A.T., Zingirian, M., Cancedda, R. & De Luca, M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349, 990–993 (1997).

    Article  CAS  Google Scholar 

  10. Tsai, R.J., Li, L.M. & Chen, J.K. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N. Engl. J. Med. 343, 86–93 (2000).

    Article  CAS  Google Scholar 

  11. Schwab, I.R., Reyes, M. & Isseroff, R.R. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea 19, 421–426 (2000).

    Article  CAS  Google Scholar 

  12. Sangwan, V.S., Vemuganti, G.K., Iftekhar, G., Bansal, A.K. & Rao, G.N. Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea 22, 478–481 (2003).

    Article  Google Scholar 

  13. Sangwan, V.S. et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J. Ophthalmol. 54, 29–34 (2006).

    Article  Google Scholar 

  14. Fatima, A. et al. Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation. J. Postgrad. Med. 52, 257–261 (2006).

    CAS  PubMed  Google Scholar 

  15. Sangwan, V.S., Fernandes, M., Bansal, A.K., Vemuganti, G.K. & Rao, G.N. Early results of penetrating keratoplasty following limbal stem cell transplantation. Indian J. Ophthalmol. 53, 31–35 (2005).

    Article  Google Scholar 

  16. Vemuganti, G.K., Kashyap, S., Sangwan, V.S. & Singh, S. Ex-vivo potential of cadaveric and fresh limbal tissues to regenerate cultured epithelium. Indian J. Ophthalmol. 52, 113–120 (2004).

    PubMed  Google Scholar 

  17. Rama, P. et al. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 72, 1478–1485 (2001).

    Article  CAS  Google Scholar 

  18. Francis, D., Abberton, K., Thompson, E. & Daniell, M. Myogel supports the ex-vivo amplification of corneal epithelial cells. Exp. Eye Res. 88, 339–346 (2009).

    Article  CAS  Google Scholar 

  19. Deshpande, P., Notara, M., Bullett, N., Daniels, J.T., Haddow, D.B. & MacNeil, S. Development of a surface-modified contact lens for the transfer of cultured limbal epithelial cells to the cornea for ocular surface diseases. Tissue Eng. Part A 15, 2889–2902 (2009).

    Article  CAS  Google Scholar 

  20. Merrett, K. et al. Tissue-engineered recombinant human collagen-based corneal substitutes for implantation: performance of type I versus type III collagen. Invest. Ophthalmol. Vis. Sci. 49, 3887–3894 (2008).

    Article  Google Scholar 

  21. Hayashida, Y. et al. Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze. Invest. Ophthalmol. Vis. Sci. 47, 552–557 (2006).

    Article  Google Scholar 

  22. Madhira, S.L., Vemuganti, G., Bhaduri, A., Gaddipati, S., Sangwan, V.S. & Ghanekar, Y. Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction. Mol. Vis. 14, 189–196 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Ang, L.P. et al. Autologous serum-derived cultivated oral epithelial transplants for severe ocular surface disease. Arch. Ophthalmol. 124, 1543–1551 (2006).

    Article  Google Scholar 

  24. Nakamura, T. et al. Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease. Ophthalmology 113, 1765–1772 (2006).

    Article  Google Scholar 

  25. Nakamura, T. et al. The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome. Invest. Ophthalmol. Vis. Sci. 47, 909–916 (2006).

    Article  Google Scholar 

  26. Kim, H.S., Jun Song, X., de Paiva, C.S., Chen, Z., Pflugfelder, S.C. & Li, D.Q. Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp. Eye Res. 79, 41–49 (2004).

    Article  CAS  Google Scholar 

  27. Tseng, S.C., Li, D.Q. & Ma, X. Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J. Cell. Physiol. 179, 325–335 (1999).

    Article  CAS  Google Scholar 

  28. Koizumi, N.J., Inatomi, T.J., Sotozono, C.J., Fullwood, N.J., Quantock, A.J. & Kinoshita, S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr. Eye Res. 20, 173–177 (2000).

    Article  CAS  Google Scholar 

  29. Ahmad, S. et al. A putative role for RHAMM/HMMR as a negative marker of stem cell-containing population of human limbal epithelial cells. Stem Cells 26, 1609–1619 (2008).

    Article  CAS  Google Scholar 

  30. Pellegrini, G. et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J. Cell. Biol. 145, 769–782 (1999).

    Article  CAS  Google Scholar 

  31. Rama, P. et al. Limbal stem-cell therapy and long-term corneal regeneration. N. Engl. J. Med. published online, doi: 10.1056/NEJMoa0905955 (23 June 2010).

Download references

Acknowledgements

We acknowledge financial support from the Hyderabad Eye Research Foundation (HERF), Sudhakar and Sreekanth Ravi (USA), the Champalimaud Foundation (Lisbon, Portugal) and from the Department of Biotechnology (DBT), Government of India. Financial support went toward all the basic and translational research efforts carried out at the S.S. Ravi Stem Cell Biology Laboratory, Prof. Brien Holden Eye Research Centre, Champalimaud Translational Centre for Eye Research (C-TRACER), L.V. Prasad Eye Institute, Hyderabad, India.

Author information

Authors and Affiliations

Authors

Contributions

D.B., G.K.V. and V.S.S. conceived the culture method and treatment option; V.S.S. carried out the biopsy and surgical transplantation; I.M., S.M., S.S., S.T., S.G. and A.F. performed the cell culture work and characterizations; I.M., S.M., S.G. and G.K.V. wrote the paper, and all authors contributed to the optimization of the protocol and the editing of the paper.

Corresponding author

Correspondence to Geeta K Vemuganti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mariappan, I., Maddileti, S., Savy, S. et al. In vitro culture and expansion of human limbal epithelial cells. Nat Protoc 5, 1470–1479 (2010). https://doi.org/10.1038/nprot.2010.115

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.115

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing