Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells
Utheim, Oeygunn (Oslo University Hospital (Oslo, Noruega))
Islam, Rakibul M (Oslo University Hospital (Oslo, Noruega))
Lyberg, Torstein (Oslo University Hospital (Oslo, Noruega))
Roald, Borghild (Oslo University Hospital (Oslo, Noruega))
Eidet, J. R (Oslo University Hospital (Oslo, Noruega))
De la Paz, Maria Fideliz (Centre d'Oftalmologia Barraquer (Barcelona, Catalunya))
Dartt, Darlene A. (Harvard Medical School)
Raeder, Sten (The Norwegian Dry Eye Clinic (Oslo, Norway))
Utheim, Tor Paaske (University of Oslo)
Universitat Autònoma de Barcelona

Data:	2015
Resum:	To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0. 04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0. 02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0. 69, P = 0. 01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0. 59, P = 0. 03). Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.
Nota:	Altres ajuts: The research was funded by the South-Eastern Norway Regional Health Authority, grant number 2010092, project name "Treating blindness using novel tissue storage technology". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Publicat a:	PloS one, Vol. 10 (march 2015) , ISSN 1932-6203

DOI: 10.1371/journal.pone.0118517
PMID: 25734654

23 p, 5.0 MB

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