To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (-183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.
- MeSH
- amnion cytologie fyziologie MeSH
- epitelové buňky cytologie fyziologie MeSH
- kryoprezervace metody MeSH
- kryoprotektivní látky MeSH
- lidé MeSH
- placenta cytologie MeSH
- rohovka cytologie fyziologie MeSH
- těhotenství MeSH
- viabilita buněk MeSH
- vitrifikace * MeSH
- zmrazování MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.
- MeSH
- apoptóza MeSH
- Descemetova membrána cytologie chirurgie MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoci rohovky chirurgie MeSH
- oční banky MeSH
- počet buněk MeSH
- retrospektivní studie MeSH
- rohovka cytologie chirurgie MeSH
- rohovkový endotel cytologie chirurgie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stroma rohovky cytologie chirurgie MeSH
- techniky tkáňových kultur metody MeSH
- zadní lamelární keratoplastika metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
PURPOSE: To assess the endothelial cell density before and after the preparation of precut posterior corneal lamellae for Descemet membrane (DM) endothelial keratoplasty with a stromal rim with the aim of standardizing this new technique and establishing the parameters of corneas intended for manual preparation under the conditions of an ocular tissue bank. METHODS: Two groups of corneoscleral buttons were used to prepare lamellae consisting of a central zone of endothelium-DM surrounded by a supporting peripheral stromal rim. Group 1 contained 12 corneas with a live endothelial cell density (LECD) of 2500 cells per square millimeter or more. Group 2 contained 10 corneas with a density of 2500 cells per square millimeter or less. The preparation was performed manually using an artificial anterior chamber. The endothelial cell density (ECD) and the percentage of dead cells were assessed before and immediately after preparation and after 48 hours of organ culture storage at 31°C. RESULTS: Immediately after preparation, on average, 4.9% and 5.2% of dead cells were found in group 1 and group 2, respectively. After 48 hours of storage, the percentage decreased significantly in both the groups. There was no significant decrease in the ECD 48 hours after preparation in group 1; however, there was a significant decrease in group 2. The amount of tissue wasted during preparation was 24%. CONCLUSIONS: The decrease in ECD is dependent on the initial values: the cell loss is less in corneas with higher original densities. We suggest that the minimal acceptable LECD of lamellae should be 2500 cells per square millimeter.
- MeSH
- buněčná smrt MeSH
- Descemetova membrána chirurgie MeSH
- endoteliální buňky cytologie patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoci rohovky patologie chirurgie MeSH
- počet buněk MeSH
- rohovkový endotel patologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stroma rohovky transplantace MeSH
- zadní lamelární keratoplastika metody MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- kryokonzervace, kapalný ethan, tekutý dusík,
- Publikační typ
- abstrakt z konference MeSH
