In vitro culture and expansion of human limbal epithelial cells

Indumathi Mariappan, Savitri Maddileti, Soumya Savy, Shubha Tiwari, Subhash Gaddipati, Anees Fatima, Virender S. Sangwan, Dorairajan Balasubramanian, Geeta K. Vemuganti

Research output: Contribution to journalArticlepeer-review

93 Scopus citations


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 42 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.

Original languageEnglish (US)
Pages (from-to)1470-1479
Number of pages10
JournalNature Protocols
Issue number8
StatePublished - Aug 2010

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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