title: In vitro culture and expansion of human limbal epithelial cells.
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 approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.
authors.affiliation: Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory, L.V. Prasad Eye Institute, Hyderabad, India.
authors.forename: Indumathi
authors.initials: I
authors.lastname: Mariappan
authors.forename: Savitri
authors.initials: S
authors.lastname: Maddileti
authors.forename: Soumya
authors.initials: S
authors.lastname: Savy
authors.forename: Shubha
authors.initials: S
authors.lastname: Tiwari
authors.forename: Subhash
authors.initials: S
authors.lastname: Gaddipati
authors.forename: Anees
authors.initials: A
authors.lastname: Fatima
authors.forename: Virender S
authors.initials: VS
authors.lastname: Sangwan
authors.forename: Dorairajan
authors.initials: D
authors.lastname: Balasubramanian
authors.forename: Geeta K
authors.initials: GK
authors.lastname: Vemuganti
mesh_terms: D000650:Amnion; D018929:Cell Culture Techniques; D003470:Culture Media; D019573:Epithelium, Corneal / Q000166:cytology*; D006801:Humans
chemical_list: D003470:Culture Media