Controlled study of the influence of storage medium type on endothelial assessment during corneal organ culture
- G Thuret1,2,
- C Manissolle1,
- S Herrag1,
- N Deb1,2,
- L Campos-Guyotat1,
- P Gain1,2,
- S Acquart3
- 1’Cell survival and adhesion in cancers and grafts,’ EA 3063, Faculty of Medicine, University Hospital of Saint-Etienne, France
- 2Ophthalmology Department, Bellevue Hospital, University Hospital, France
- 3Cornea Bank/French Blood Centre, Saint-Etienne, France
- Correspondence to: Gilles Thuret Service d’Ophtalmologie, Pavillon 50A, Hôpital Bellevue, CHU Saint-Etienne, 42055 Saint-Etienne Cedex 2, France; gilles.thuretuniv-st-etienne.fr
- Accepted 26 May 2003
Selection of corneal grafts in eye banks is mainly based on end-of-storage endothelial assessment, which consists of endothelial cell density (ECD) measurement and, to some extent, cell morphometry. Below a certain ECD threshold, generally 2000 cells/mm2, the cornea is deemed unfit for penetrating keratoplasty. Precise ECD measurement at the end of storage is thus a key issue for eye banks, and also for patients, because it influences the long term survival of the graft.1–3
For long term storage in organ culture, the most common method in Europe,4 endothelial observation is possible only by transmitted light microscopy. The endothelial cells are exposed to 0.9% sodium chloride or sometimes to 1.8% sucrose, which induce a small degree of osmotic cell shrinkage and dilatation of the intercellular spaces thus making individual cells visible. The cells can then be counted manually, through a calibrated reticule or from a photograph; or using an advanced image analysis system.5–7 Whichever method of count is used, precision depends primarily on good visualisation of the cell borders. It has long since been shown that, even under experimental conditions of perfect cell membrane visualisation using alizarin red, maximum precision ranges from +5% to −5%.8
Two commercial media are authorised by the Agence Française de Sécurité Sanitaire des Produits de Santé. They are very similar in composition, both being based on HEPES-buffered Iscove’s Modified Dulbecco’s medium containing sodium bicarbonate and 2% fetal bovine serum, with the same pH of 7.25 but the osmolality of Inosol (Bausch & Lomb, Chauvin-Opsia, Labège, France) is only 305 mosmol/kg (range 295–315) compared with 320 mosmol/kg (range 300–340) for CorneaPrep/Max (Eurobio, Les Ulis, France). One has nevertheless acquired the reputation of allowing better visualisation of endothelial cells and thus facilitating ECD measurement. We therefore compared the quality of …
