Dataset Information

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.

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

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Publications

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.

Utheim Oeygunn O Islam Rakibul R Lyberg Torstein T Roald Borghild B Eidet Jon Roger JR de la Paz Maria Fideliz MF Dartt Darlene A DA Raeder Sten S Utheim Tor Paaske TP

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]

PMID: 25734654

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Project description:To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA).Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay.TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus.These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

Dataset Information

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.

Aim/purpose of the study

Materials and methods

Results

Conclusion

Publications

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.

Similar Datasets

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