[Corneal cryopreservation in man: a proposal for an original technic].
Using the method of Capella and Kaufman, the authors propose an original cryopreservation technique of human corneas. Sixty biomicroscopically normal corneas were obtained from cadavers (50-70 years) within six hours after death. Initial protocol involves four phases: 1) cryoprotection is carried out in three baths of three minutes each with human plasma and DMSO (dimethylsulfoxide) at 2-4-7%; 2) freezing is performed with 10 ml of the final cryoprotective fluid in a controlled rate freezer, the "Minicool" (CFPO). The cornea is cooled at a rate of 2 degrees C/min from + 6 degrees C down - 40 degrees C then at 5 degrees C/min down - 140 degrees C; 3) a tank containing liquid nitrogen at - 150 degrees C is used for a long term storage; 4) thawing is carried out in a water-bath at 37 degrees C for one minute, then in serum albumin 25% for three minutes and in M-K medium for thirty minutes. Effects of three cryobiological parameters are studied: cryoprotection: DMSO is diluted with human plasma at the following increasing concentrations: 2-4-7% or 4-6-10%; freezing medium: the cornea is frozen with or without the final cryoprotective bath. Thus freezing is carried out either in dry or liquid phase; thawing: initial temperature and the osmolar decrease of the dilution are modified in order to obtain four protocols: n degrees 1, initial protocol; n degree 2, an additional step using Hanks 5% - SA 25% for three minutes (1 000 mosm); n degree 3, after initial step reheating the cornea is directly placed in M-K medium; n degree 4, temperature of 50 degrees C for ten seconds precedes the initial protocol. For each protocol change, the endothelial viability is controlled by histological and/or SEM studies results: For each protocol change, the endothelial viability is controlled by histological and/or SEM studies results: cellular osmotic lesions are greater for DMSO concentrations of 10%; dry phase freezing induces more intense and more diffuse nuclear damage than in liquid phase; thawing under protocols 4 and 2 cause a high degree of nuclear damage associated with diffuse cytoplasmic vacuole formation. Histological and SEM studies clearly show endothelial cell lesions due to the cryoconservation. The human cornea endothelium is affected by any modification of the cryobiological parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
D000368:Aged; D003315:Cornea / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004727:Endothelium / Q000473:pathology / Q000648:ultrastructure; D005615:Freezing; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009926:Organ Preservation / Q000379:methods*; D010949:Plasma; D014022:Tissue Survival
D004121:Dimethyl Sulfoxide
La cryoconservation cornéenne chez l'homme: proposition technique originale.