Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome.
OBJECTIVE
To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system.


METHODS
Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4 degrees C in an eye bank corneal storage medium. Differences were tested using nonparametric methods.


METHODS
The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium.


RESULTS
At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4 degrees C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7-100%) versus 98.4% (range, 88.9-100%) (P>0.05); on day 3, 100% (range, 64.3-100%) versus 63.4% (range, 13.6-95.5%) (P<0.0005); on day 6, 95.0% (range, 35.0-100%) versus 28.0% (range, 0-92.0%) (P<0.0005); and on day 9, 95.0% (range, 3.7-100%) versus 68.6% (range, 0-100%) (P<0.0005).


CONCLUSIONS
After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.
Tungsiripat
Tulaya
T
Department of Ophthalmology, University of California-Irvine, 118 Med Surge I, Irvine, CA 92697-4375, USA.
Sarayba
Melvin A
MA
Taban
Mehran
M
Sweet
Paula M
PM
Osann
Kathryn E
KE
Chuck
Roy S
RS
D002452:Cell Count; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation; D019573:Epithelium, Corneal / Q000166:cytology* / Q000502:physiology / Q000637:transplantation; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D018613:Microscopy, Confocal; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D014019:Tissue Donors; D014021:Tissue Preservation; D020858:Tissue and Organ Harvesting