1. EPITHELIAL DEFECTS IN CORNEAS IN ORGAN CULTURE CTS-Manchester Eye Bank & Ophthalmic Pathology Service Isaac Zambrano Fiona Carley Luciane Irion Richard Bonshek

2. Taking the eye out of the pot Cleaning the remains of the tissue Washing eyes in saline, iodine & Sodium thiosulphate Cutting sclera with trephine Excision of corneoscleral disc using scalpel Inserting suture in sclera Cleaning of eyes and excision of corneas

3. Cornea - organ culture (31 to 37°C) Culture medium: Supply electrolytes, nutrients, vitamins, hormones, proteins Culture medium: Supply electrolytes, nutrients, vitamins, hormones, proteins Maintain metabolism Maintain metabolism Support cell proliferation and protein synthesis Support cell proliferation and protein synthesis Support cellular repair mechanisms Support cellular repair mechanisms

4. Placing corneas in organ culture Placing the corneoscleral disc in organ culture Incubating for up to 28 days at 34 0 C

5. Issuing corneas Release from quarantine Release from quarantine 3 days before scheduled date of graft, corneal endothelium examined by light microscopy 3 days before scheduled date of graft, corneal endothelium examined by light microscopy Estimate cell density (minimum 2200 cells/mm²) Estimate cell density (minimum 2200 cells/mm²) Dead/missing cells (trypan blue staining) Dead/missing cells (trypan blue staining) Opacities Opacities If suitable for PK, cornea placed into medium containing 5% dextran T500 to reverse stromal oedema and returned to 34°C If suitable for PK, cornea placed into medium containing 5% dextran T500 to reverse stromal oedema and returned to 34°C Cornea despatched at ambient temperature to recipient hospital day before graft

6. 3 Cases Corneas stored in culture Epithelial defects before or after culture with dextran medium

7. FCY-13A. 75 years old male. Death to enucleation: 15hrs Enucleation to culture: 17hrs Death to culture: 33hrs Cause of death: Pneumonia, Myocardial infarction

8. FCY13-A

9. FCY-13A

10. FCY-3A 60 years old male. Death to enucleation: 20hrs Enucleation to culture: 8hrs Death to culture: 28hrs Cause of death: alcoholic liver disease

11. FCY-3A

15. FCY-1B 60 years old female Death to donation: 19hrs Enucleation to culture: 18hrs Death to culture: 38hrs Cause of death: Ca Lung

16. FCY1-A

17. FCY1-B

18. FCY1

20. SUMMARY Several cases of epithelial defects in corneas stored in organ culture Often visible only when corneas are transferred to culture medium with dextran Epithelial defects are of non-infectious nature No correlation with time of death to enucleation and to storage, age, endothelial cell count or cause of death Why some corneas develop such abnormalities?