A novel method for preserving cultured limbal epithelial cells.
OBJECTIVE
To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue-engineered epithelia that are used to treat patients with limbal stem cell deficiency.


METHODS
Limbal epithelial cells were cultured for 3 weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1 week at 23 degrees C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry.


RESULTS
The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non-preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained.


CONCLUSIONS
Cultured limbal epithelial cells can be preserved in organ culture medium for 1 week at room temperature, while maintaining the original layered structure and undifferentiated phenotype.
Utheim
Tor Paaske
TP
Department of Ophthalmology, Centre for Eye Research, University of Oslo, Ullevål University Hospital, Kirkeveien 166, 0407 Oslo, Norway.
Raeder
Sten
S
Utheim
Øygunn Aass
ØA
Cai
Yiqing
Y
Roald
Borghild
B
Drolsum
Liv
L
Lyberg
Torstein
T
Nicolaissen
Bjørn
B
D000650:Amnion; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D003470:Culture Media; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D013696:Temperature
D003470:Culture Media