Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation.
OBJECTIVE
To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage.


METHODS
HLEC cultured on amniotic membranes were stored in organ culture medium in a closed container at 23 degrees C. Sterility of storage media was tested using a Bactec 9240 blood culture instrument (Becton Dickinson, Maryland) for incubation and periodic reading. Viability was analysed by calcein-acetoxymethyl ester/ethidium homodimer-1 assay, morphology by light microscopy and cellular phenotype by immunohistochemistry.


RESULTS
No microbial contamination was observed after 1 week's storage. Viability of cultured HLEC was 87.9 (SD 6.4)% and 52.7 (13.1)% after 2 and 3 weeks of storage, respectively, compared with 98.8 (2.6)% before storage (p<0.001). The multilayered structure was preserved in 70% of cultures following 2 weeks of storage but lost after 3 weeks. A less differentiated phenotype was maintained.


CONCLUSIONS
This study is the first to verify the sterility of HLEC cultures prior to transplantation. Although a slight decrease in viability was observed following 2 weeks of storage, the HLEC sheets remain acceptable, whereas 3 week's storage was unsatisfactory.
Utheim
T P
TP
Center for Clinical Research, University of Oslo, Oslo, Norway.
Raeder
S
S
Utheim
Ø A
ØA
de la Paz
M
M
Roald
B
B
Lyberg
T
T
D002478:Cells, Cultured / Q000166:cytology* / Q000637:transplantation; D003316:Corneal Diseases / Q000473:pathology; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology* / Q000637:transplantation; D019573:Epithelium, Corneal / Q000166:cytology*; D005125:Eye Banks; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D009926:Organ Preservation; D013194:Staining and Labeling; D033581:Stem Cell Transplantation; D013242:Sterilization; D014022:Tissue Survival
D003470:Culture Media