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


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 (ΔNp63α, 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 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).


CONCLUSIONS
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.
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Department of Ophthalmology, Oslo University Hospital, Oslo, Norway.
Oeygunn
O
Utheim
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
Rakibul
R
Islam
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Torstein
T
Lyberg
Department of Pathology, Oslo University Hospital, Oslo, Norway.
Borghild
B
Roald
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Jon Roger
JR
Eidet
Institut Universitari Barraquer, Universitat Autonoma de Barcelona, Barcelona, Spain.
Maria Fideliz
MF
de la Paz
Schepens Eye Research Institute / Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States of America.
Darlene A
DA
Dartt
The Norwegian Dry Eye Clinic, Oslo/Tromsoe, Norway.
Sten
S
Raeder
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway; Department of Ophthalmology, Oslo University Hospital, Oslo, Norway; Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
Tor Paaske
TP
Utheim
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000650:Amnion / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D051743:CCAAT-Enhancer-Binding Protein-delta / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002470:Cell Survival; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D053539:Keratin-19 / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D048054:Mitogen-Activated Protein Kinase 7 / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; C497252:CEBPD protein, human; C081876:CKAP4 protein, human; D018031:Connexin 43; D053539:Keratin-19; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D051743:CCAAT-Enhancer-Binding Protein-delta; C481581:MAPK7 protein, human; D048054:Mitogen-Activated Protein Kinase 7; D053148:Caspase 3