Journal Article

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Islam, Rakibul, Dartt, Darlene A., Eidet, Jon Roger, Utheim, Oeygunn, de la Paz, Maria Fideliz, Lyberg, Torstein, Roald, Borghild, Raeder, Sten, Utheim, Tor Paaske

2015

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amnion - cytology / Amnion - metabolism / Amniotic membrane / Analysis / Apoptosis / ATP Binding Cassette Transporter, Sub-Family G, Member 2 / ATP-Binding Cassette Transporters - genetics / ATP-Binding Cassette Transporters - metabolism / Biochemistry / Biomarkers - metabolism / Bmi protein / Caspase / Caspase 3 - genetics / Caspase 3 - metabolism / Caspase-3 / CCAAT-Enhancer-Binding Protein-delta - genetics / CCAAT-Enhancer-Binding Protein-delta - metabolism / CCAAT/enhancer-binding protein / Cell Differentiation / Cell proliferation / Cell Survival / Connexin 43 / Connexin 43 - genetics / Connexin 43 - metabolism / Containers / Cytology / Dentistry / Epithelial cells / Epithelial Cells - cytology / Epithelial Cells - metabolism / Explants / Gene Expression / Genotype & phenotype / Hospitals / Humans / Keratin-19 - genetics / Keratin-19 - metabolism / Limbus Corneae - cytology / Limbus Corneae - metabolism / Markers / Medical research / Membrane Proteins - genetics / Membrane Proteins - metabolism / Membranes / Mitogen-Activated Protein Kinase 7 - genetics / Mitogen-Activated Protein Kinase 7 - metabolism / Morphology / Neoplasm Proteins - genetics / Neoplasm Proteins - metabolism / Preservation / Primary Cell Culture / Proliferating cell nuclear antigen / Proliferating Cell Nuclear Antigen - genetics / Proliferating Cell Nuclear Antigen - metabolism / Serum-free medium / Sheets / Stem cell transplantation / Stem cells / Storage / Surgery / Tissue Culture Techniques - methods / Transplants & implants / Ultrastructure / Xenobiotics