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