Dataset Information
Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.
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ABSTRACT: Aim/purpose of the study
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.Materials and methods
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 (?Np63?, p63, Bmi-1, C/EBP?, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3).Results
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 ?m, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ?Np63? 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 ?Np63?, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03).Conclusion
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 ?Np63?-positivity after both 4 and 7 days.
SUBMITTER: Utheim O
PROVIDER: S-EPMC4348416 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.
PloS one 20150303 3
<h4>Aim/purpose of the study</h4>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.<h4>Materials and methods</h4>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 m ...[more]