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.
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Acknowledgement
We thank Fondazione Roma and Colin Murdoch for their generous help. The corresponding author personally dedicates this chapter to all goodwill Italian researchers, whose contribution is still vital for the country in such difficult times. This manuscript was financially supported by University of Rome “Sapienza,” Grant “Ateneo 2011 Prot. C26A11MP7J,” Department of Science and Medical-Surgical Biotechnologies, University of Rome “Sapienza,” Polo Pontino, Latina, Italy.
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Scafetta, G., Siciliano, C., Frati, G., De Falco, E. (2014). Culture of Human Limbal Epithelial Stem Cells on Tenon’s Fibroblast Feeder-Layers: A Translational Approach. In: Turksen, K. (eds) Stem Cells and Good Manufacturing Practices. Methods in Molecular Biology, vol 1283. Humana Press, New York, NY. https://doi.org/10.1007/7651_2014_102
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DOI: https://doi.org/10.1007/7651_2014_102
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