Effects of organ culture and Optisol-GS storage on structural integrity, phenotypes, and apoptosis in cultured corneal epithelium.
OBJECTIVE
A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared.


METHODS
Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).


RESULTS
The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low.


CONCLUSIONS
These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.
Center for Eye Research, Department of Ophthalmology, Ulleval University Hospital, University of Oslo, Norway. sten.rader@medisin.uio.no
Sten
S
Raeder
Tor P
TP
Utheim
Oygunn A
OA
Utheim
Bjørn
B
Nicolaissen
Borghild
B
Roald
Yiqing
Y
Cai
Kristiane
K
Haug
Anders
A
Kvalheim
Edvard B
EB
Messelt
Liv
L
Drolsum
John C
JC
Reed
Torstein
T
Lyberg
D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D002809:Chondroitin Sulfates; D045424:Complex Mixtures; D015925:Cryopreservation / Q000379:methods*; D016895:Culture Media, Serum-Free; D003911:Dextrans; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D019573:Epithelium, Corneal; D005125:Eye Banks; D005839:Gentamicins; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D016850:Limbus Corneae / Q000166:cytology*; D046529:Microscopy, Electron, Transmission; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques; D009926:Organ Preservation / Q000379:methods*; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers; D045424:Complex Mixtures; D016895:Culture Media, Serum-Free; D003911:Dextrans; D005839:Gentamicins; D002809:Chondroitin Sulfates; C075736:Optisol; D053148:Caspase 3