[Eye banks and available transplants]. / Biobank for øyesykdommer.

BACKGROUND: Eye banks have procured, processed and stored donor corneas for decades. In parallel, new techniques have emerged employing allogeneic transplantation of various cells and tissues from the eye banks. This progress is a consequence of increased knowledge of stem cells, cell kinetics and immunological aspects and improved techniques for cell culturing, tissue storage and microsurgery. MATERIAL AND METHODS: Review article on available transplants for treating eye diseases, based on experience with eye banking, clinical ophthalmological practice, own research and literature retrieved from PubMed, Medline and www.google.com. RESULTS AND INTERPRETATION: Treatment techniques for eye diseases, which require biological material for grafting, need efficient eye banks for continuous supply of donor material of high quality. New Norwegian legislation, based on implementation of EU Directive 2004/23/EC, demands authorization of all eye banks. The EU Directive sets high and rigorous standards for quality and safety for donation, procurement, testing, processing, storage and distribution of tissues and cells. Well-run eye banks are of great importance for modern treatment of patients suffering from eye diseases and for progress and research in ophthalmology.

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.

PURPOSE: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. METHODS: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. RESULTS: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. CONCLUSIONS: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.

Regeneration of the epithelium in organ-cultured donor corneas with extended post-mortem time.

PURPOSE: The maximum post-mortem time limit for obtaining donor corneas varies between eye banks. It is not known for how long a time the epithelial cells survive post-mortem, nor is it known if donor corneas with extended post-mortem time are able to regenerate the epithelium. Therefore, we wanted to examine the epithelium in donor corneas for regenerative ability during storage in an eye bank organ culture system. METHODS: Twenty-four paired donor corneas with post-mortem time from 28 to 163 hr were obtained. One cornea of a pair was fixed immediately to serve as a control, and the second was cultured in eye bank medium at 32 degrees C for 3 days. Examination of the specimens was performed with light and scanning electron microscopy. Immunohistochemical staining methods with antibodies against K 3, K 19, vimentin and p63 were used to further characterize the cells. RESULTS: The control corneas showed decreasing amounts of epithelial cells with increasing post-mortem time. All the cultured corneas demonstrated rapid regeneration of the epithelium. After 3 days in organ culture, 10 of 12 donor corneas were covered with epithelium. CONCLUSION: Even up to 7 days post-mortem, viable cells reside in the corneal epithelium. The study demonstrates the hardiness and enormous regenerative potential of peripheral corneal cells. Donor corneas processed in an eye bank organ culture storage system will obtain an intact epithelial layer within a few days.

Effect of limbal explant orientation on the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells.

PURPOSE: To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols. METHODS: Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium (n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of DeltaNp63alpha, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy. RESULTS: The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 +/- 4.2%) than the stromal group (75.3 +/- 8.9%), and Western blots showed a stronger band of DeltaNp63alpha. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells. CONCLUSIONS: Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and DeltaNp63alpha.

Donor corneas for transplantation: a scanning electron microscopic study of the epithelium.

PURPOSE: Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. METHODS: A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. RESULTS: Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. CONCLUSION: The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Rapid determination of polyether marine toxins using liquid chromatography-multiple tandem mass spectrometry.

The diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxins (DTX); pectenotoxin-2 (PTX2) and pectenotoxin-2 seco acids, were determined in marine phytoplankton, Dinophysis acuta, and mussels (Mytilus edulis) collected along the southwest coast of Ireland. Liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) was employed for the simultaneous determination of a series of marine toxins with large polarity differences. Separation of five DSP toxins was achieved on a C18 column (Luna-2, 150 mm x 2.1 mm, 5 microm) using an acetonitrile-water gradient with ammonium acetate as an eluent modifier. Electrospray ionisation (ESI) in negative mode, was used to generate the molecule related ion, [M-H]-, for each toxin. To develop a multiple reaction monitoring (MRM) method, fragmentation studies were performed to determine the optimum precursor-product ion combinations: OA (803/255), DTX2 (803/255), DTX1 (817/255), PTX2SAs (875/137) and PTX2 (857/137). This highly sensitive method had detection limits better than 1 pg (on-column). Linear calibrations were obtained for shellfish extracts that were spiked with toxins, OA, 0.007-1.00 microg/ml (r2 = 0.9993, N = 3) and DTX2, 0.054-8.5 microg/ml (r2 = 0.9992, N = 3). Good reproducibility data were also achieved with %RSD values (N = 3) ranging from 3.15% (0.56 microg DTX2/ml) to 5.71% (0.14 microg DTX2/ml), for shellfish extracts. The method was sufficiently sensitive to permit the determination of DSP toxins in small numbers of picked phytoplankton cells (N = 12-40). In one sample of D. acuta the average toxin composition per cell was: OA (7.0 pg), DTX2 (11 pg) and PTX2 (7.2 pg).

First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish.

Azaspiracids have recently been identified as the toxins responsible for a series of human intoxications in Europe since 1995, following the consumption of cultured mussels (Mytilus edulis) from the west coast of Ireland. Liquid chromatography-mass spectrometric (LC-MS) methods have been applied in the study reported here to investigate the new human toxic syndrome, azaspiracid poisoning. Separation of azaspiracid (AZA1) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. These azaspiracids have now been identified in mussels from Craster (north-east England) and Sognefjord (south-west Norway) using source collision induced dissociation-MS and multiple tandem MS detection. AZA1 was the predominant toxin and toxin profiles were similar to those found in contaminated Irish shellfish. This is the first report of the occurrence of these azaspiracids outside Ireland with the significant implications that these toxins may occur in shellfish throughout northern Europe.