Ex-vivo potential of cadaveric and fresh limbal tissues to regenerate cultured epithelium.
OBJECTIVE
To evaluate and compare the ex-vivo growth potential and formation of cultured corneal epithelium from residual corneo-limbal rings obtained from the operating room after penetrating keratoplasty, and fresh limbal tissues from patients undergoing routine cataract surgery.


METHODS
With the approval of the Institutional Review Board and informed consent from patients, 1-2 mm of limbal tissues from 15 patients and 31 tissues from the cadaveric limbal ring preserved in MK medium (16 tissues) and Optisol (15 tissues) were used for the study. Donor data included age, time lapse between death and collection, collection and preservation and preservation and culture. Tiny bits of the limbal tissue were explanted on the de-epithelialised human amniotic membrane prepared following standard guidelines, and cultured using Human Corneal Epithelial cell medium. Radial growth from the explant was observed and measured by phase contrast microscopy over 2-4 weeks. After adequate confluent growth, whole mount preparation of the membrane was made and stained with haematoxylin and eosin. Part of the membrane was fixed in formalin and processed for routine histologic examination. The sections were stained with haematoxylin and eosin.


RESULTS
Forty-six tissues were evaluated from 42 eyes (15 from patients, 31 from cadaveric eyes) with a mean age of 55.3 years +/- 21.23 years (range 18 years - 110 years). The growth pattern observed was similar in all the positive cases with clusters of cells budding from the explant over 24-72 hours, and subsequent formation of a monolayer over the next 2-3 weeks. The stained whole mount preparation showed a radial growth of cells around explants with diameter ranging from 5 to 16mm. Histologic evaluation of the membrane confirmed the growth of 2-3 cell-layered epithelium over the amniotic membrane. Cultivated epithelium around explant cell cultures was observed in 100% (15/15) of limbal tissue obtained from patients, as against 56% (9/16) of MK medium preserved tissues and 46.7% (7/15) of Optisol preserved tissues. This was statistically significant (P=0.0131) There was no significant statistical difference in the growth properties, i.e, the mean percentage of fragments showing growth (P=0.229) or the mean diameter of growth (P=0.479) in the cultures obtained from fresh and preserved tissues. The time lapse at various stages between death and utilisation and donor age had no significant influence on the growth potential of the limbal tissues.


CONCLUSIONS
The potential for generating cultured corneal epithelium from fresh limbal tissues obtained from living subjects is higher than that observed with preserved tissues. It would also be worthwhile to address the factors that could further enhance the proliferative potential of the cadaveric tissues obtained from eye banks.
Department of Ophthalmic Pathology Service, L V Prasad Eye Institute, Hyderabad, India. geeta@lvpei.org
Geeta K
GK
Vemuganti
Subhadra
S
Kashyap
Virender S
VS
Sangwan
Shashi
S
Singh
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000650:Amnion; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000166:cytology* / Q000502:physiology; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D008875:Middle Aged; D012038:Regeneration; D014019:Tissue Donors