Protocol abstract


Nature Protocols 5, 1470 - 1479 (2010)
Published online: 29 July 2010 | doi:10.1038/nprot.2010.115

Subject Categories: Cell and tissue culture | Model organisms | | | |

In vitro culture and expansion of human limbal epithelial cells

Indumathi Mariappan1, Savitri Maddileti1, Soumya Savy1, Shubha Tiwari1, Subhash Gaddipati1, Anees Fatima2, Virender S Sangwan3, Dorairajan Balasubramanian4 & Geeta K Vemuganti1,5


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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  1. Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Hyderabad, India.
  2. Department of Dermatology, The Feinberg School of Medicine, Northwestern University, Chicago, USA.
  3. Cornea and Anterior Segment Services, L.V. Prasad Eye Institute, Hyderabad, India.
  4. Prof. Brien Holden Eye Research Centre, C-TRACER, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India.
  5. Ophthalmic Pathology Services, L.V. Prasad Eye Institute, Hyderabad, India.

Correspondence to: Geeta K Vemuganti1,5 e-mail: geeta@lvpei.org

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