title: Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.
abstract: The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.
authors: Department of Medical-Surgical Science and Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome "Sapienza", C.so della Repubblica 79, 04100, Latina, Italy.
authors: Gaia
authors: G
authors: Scafetta
authors: Camilla
authors: C
authors: Siciliano
authors: Giacomo
authors: G
authors: Frati
authors: Elena
authors: E
authors: De Falco
mesh_terms: D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D018920:Coculture Techniques / Q000379:methods*; D003114:Colony-Forming Units Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism