Michael Hagenah

Incidence of bacterial and fungal contamination of donor corneas preserved by organ culture.

We reviewed the results of sterility testing from culture media of 1,134 donor corneas preserved by organ culture at 37°C in our eye bank. All corneas were stored in minimal essential medium containing 2% fetal calf serum, 0.1 mg/ml penicillin G, 0.1 mg/ml streptomycin, and 2.5 u.g/ ml amphotericin B. After removal of ocular adnexal tissue, donor globes were rinsed with sterile saline solution, incubated in 3% polyvinylpyrrolidone-iodine solution for 3-5 min, and subsequently rinsed again with sterile saline solution. Samples for microbiological evaluation were obtained from the initial evaluation medium, at every medium change (every 10 days), and from the medium used for deswelling of the individual cornea 1 day before transplantation. Incidence of contamination was 0.53% (6 of 1,134 corneas). Three corneas were contaminated by Micrococcus species, three by fungi. We conclude from our study that a combination of rinsing donor globes with sterile saline solution, the initial use of a disinfectant, and the employment of penicillin/streptomycin and amphotericin B in the organ culture medium, which have been commonly used in cell culture for decades, result in a low incidence of bacterial and fungal contamination of corneas preserved by organ culture.

Impact of transportation on short-term preserved corneas preserved in Optisol-GS, Likorol, Likorol-DX, and MK-medium.

Purpose. To investigate the impact of transportation, simulated under laboratory conditions, on the corneal endothelium preserved by Optisol-GS, Likorol-DX, Likorol, and MK-Medium. Methods. Three hundred twenty corneas from freshly slaughtered pigs were stored in Optisol-GS, Likorol-DX, Likorol, and MK-Medium for 1, 3, 6, or 10 days. After short-term preservation, the transportation was simulated on a laboratory shaker at 4°C with an acceleration of 0–100 km/h in 16 seconds for 5 hours. Mate corneas served as the control. Corneal endothelial cell density was determined at the time of dissection, directly before the experiment, and after subsequent organ culture. Results. A significant cell loss induced by transportation simulation was not observed in any experimental group. Preservation in MK-Medium starting at 3 days of short-term preservation resulted in a significant cell loss. Storage for up to 6 days in Optisol-GS, Likorol-DX, and Likorol did not result in a significant decrease in cell density. Conclusion. Short-term preserved corneas can be routinely distributed without a reevaluation, if the tissue is preserved for a shorter time as recommended by the manufacturers.

Entwicklung der Endothelzelldichte bei Verwendung von frischem und organkultiviertem Gewebe 5 Jahre nach perforierender Keratoplastik

Viele klinische Studien befaßten sich mit der Reduktion der Endothelzelldichte nach perforierender Keratoplastik bei Verwendung unterschiedlicher Konservierungsmethoden des Spendergewebes. Problematisch war hier meist die Deutung der Ergebnisse, da trotz seit langem bekanntem dynamischem Zellverlust sehr variable Nachuntersuchungsintervalle zusammengefaßt wurden. Das Ziel dieser Untersuchung war es, unter Berücksichtigung 2er verschiedener Konservierungstechniken eine langfristige Endothelbefundung zu gleichen Terminen durchzuführen.54 Patienten wurden 5,2 (±0,5) bzw. 4,9 (±0,6) Jahre nach perforierender Keratoplastik nachuntersucht. 24 Patienten hatten eine frisch vom Bulbus präparierte, 30 Patienten eine organkultivierte Hornhaut erhalten. Alle Hornhäute waren im Nachbeobachtungszeitraum klar gelieben.Die Nachbeobachtungszeit unterschied sich nicht signifikant (p = 0,26). Unterschieden sich sowohl Post-mortem-Zeit (p<0,001) zugunsten frisch transplantierten Gewebes und Zelldichte vor der Operation (p<0,001) zugunsten organkultivierten Gewebes signifikant, konnten solche Differenzen für die Variablen Spenderalter (p = 0,64) und Patientenalter (p = 0,46) nicht gefunden werden. Die durchschnittliche Endothelzelldichte war nach 5 Jahren mit 1070 (±499) Zellen/mm2 bei Verwendung frisch transplantierten Gewebes und 1095 (±497) Zellen/mm2 bei Verwendung organkultivierter Hornhäute nicht signifikant verschieden (p = 0,82).Bei Verwendung der Variablen Endothelzelldichte ließen sich 5 Jahre nach perforierender Keratoplastik keine Unterschiede zwischen beiden Konservierungstechniken feststellen. Wir bevorzugen die Organkultur, da neben der deutlich geringeren Inzidenz primären Transplantatversagens auch Spendergewebe mit längeren Post-mortem-Zeiten in einem planbaren Eingriff eingesetzt werden kann.Many clinical studies have been performed investigating corneal endothelial cell loss after penetrating keratoplasty using different preservation methods. Most studies, however, included variable follow-up intervals, neglecting the dynamic cell loss over the course of time. The aim of this study was to perform endothelial cell evaluation 5 years after penetrating keratoplasty using two different storage methods.Fifty-four patients were examined 5.2 (±0.5) or 4.9 (±0.6) years after surgery. Twenty-four patients had received a cornea stored in a moist chamber, 30 patients a cornea preserved by organ culture. All corneas had remained clear in the follow-up period.The follow-up periods did not differ significantly (P = 0.26). The post-mortem time of moist-chamber-stored tissue was significantly lower (P<0.001), endothelial cell density significantly higher (P<0.001) in organ-culture-preserved corneas. Donor age (P = 0.64) and patient age (P = 0.046) did not differ significantly. Average corneal endothelial cell density was 1070 (±499) cells/mm2 using moist chamber stored and 1095 (±497) cells/mm2 using organ-culture-preserved tissue (P = 0.82).Five years after penetrating keratoplasty, we were not able to find significant differences in corneal endothelial cell density using either moist chamber or organ-culture-preserved corneas. We prefer to use organ culture since there is a lower incidence of primary graft failure, tissue with longer post-mortem times can be used, and surgery can be scheduled.

Latent endothelial cell damage after experimental corneal cryopreservation

Ninety porcine corneas were evaluated by vital staining with alizarin red S and trypan blue in a three-step experiment. Central cell densities were counted (a) on freshly dissected corneas (n = 30), (b) on cryopreserved corneas directly after thawing (n = 30), and (c) after a postthawing organ culture interval of 24 h (n = 30). Two freezing methods were used: (a) minimum essential medium — containing 20% fetal calf serum and (b) the same but containing additionally 2% chondroitin sulfate. Directly after thawing neither method showed significant cell loss (3.9% and 3%) compared to fresh tissue. After postthawing organ culture, however, tissue that had been frozen without chondroitin sulfate displayed a cell loss of 73.5% compared to corneas of the same freezing protocol directly after thawing. Corneas in chondroitin sulfate containing medium showed a cell loss of only 33.2%. We conclude that reliable morphologic evaluation should not be obtained from cryopreserved corneas examined directly after thawing.

Endothelial cell death in organ-cultured donor corneae : the influence of traumatic versus nontraumatic cause of death

Abstract• Background: Donors who have suffered a traumatic death are, on average, younger than those who have died from other causes, and the time from death to enucleation (DET) also tends to be shorter. Corneae obtained from such donors are therefore considered particularly suitable for grafting. One of the reasons for excluding a donor cornea from transplantation is the occurrence of endothelial cell necrosis during organ culture, and we investigated whether the incidence of this phenomenon bears a relationship to death by traumatic or nontraumatic means. • Methods: Data from 2125 donor corneae were collected using standardized evaluation protocols between January 1991 and December 1995 and included information on cause of death, age and DET, as well as endothelial cell loss and necrosis. Traumatic deaths were recorded in 346 cases, nontraumatic deaths in the other 1779 cases. Since differences in age (P = 0.006) but not in DET occurred within each of these groups, a more refined comparison, with matched data (< 35 years), was also undertaken. • Results: Forty (11.6%) of the 346 corneae derived from traumatic death donors manifested total or partial endothelial cell death in organ culture. The corresponding figure in the nontraumatic death group was only 105/1779 (5.8%;P = 0.0002). After matching for age, endothelial cell death during culturing was revealed in 18 (13.5%) of the 133 of the traumatic death corneae and in 3 (2.6%) of the 115 nontraumatic death ones (P = 0.004); the overall incidence of endothelial cell death (total or partial) during organ culture was 6.8% (145/2125). Endothelial cell loss during culturing of the 227 age-matched donor corneae which still had an intact endothelial monolayer at the end of the incubation period was 340 ± 388 cells/mm2 in traumatic death corneae (n = 115) and 255 ± 318 cells/mm2 in nontraumatic death ones (n = 112;P = 0.051). • Conclusion: Corneae obtained from traumatic death donors were more liable to undergo total or partial endothelial cell death during organ culture than were those procured from nontraumatic death ones. However, in corneae which survived the period of culture, there was no significant difference in endothelial cell loss between the two groups. Whilst the mechanism underlying this increased susceptibility of traumatic death corneae to cell death remains elusive, the data gleaned from this investigation nonetheless emphasize the potential importance of being able to perform meaningful in vitro viability tests on donor corneae; this is possible only under organ culture conditions.

Experimental corneal cryopreservation : impact of postmortem time on corneal endothelial cell survival

Clinical and experimental studies with rabbit and human corneas have shown the correlation between short postmortem times and successful corneal cryopreservation. In this experimental study we investigated this phenomenon considering the latent freeze-thaw-induced cell damage. Enucleated eye-balls of freshly slaughtered pigs were stored in moist chambers at 4 degrees C for 2, 4, 8, 16, 32, and 72 h before cryopreservation. After thawing, the corneas were organ-cultured for 1 day. After staining with trypan blue and alizarin red S the tissue was evaluated morphometrically, calculating the amount of necrotic areas on the central corneal surface and the endothelial cell density. Corneas stored up to 32 h before cryopreservation showed no difference regarding the amount of necrosis and endothelial cell density compared to freshly cryopreserved tissue. Corneas stored 72 h before cryopreservation revealed no endothelial cell survival. We conclude that a post-mortem time of up to 32 h before corneal cryopreservation has no influence on endothelial cell survival.

Entwicklung der Endothelzelldichte von Hornhäuten mit langen Post-Mortem-Zeiten bei Kurzzeitkonservierung mit Optisol

ZusammenfassungFür die Bereitstellung von Spendergewebe für die perforierende Keratoplastik ist die Kurzzeitkultur eine Methode, das Gewebe mit speziellen Kulturmedien und geringem apparativen Aufwand zu konservieren. Es wird jedoch empfohlen, nur Hornhäute mit einer post-mortem Zeit von weniger als 6 Stunden zu verwenden. In einer experimentellen Studie wurde diese Empfehlung überprüft. Bulbi frisch geschlachteter Schweine wurden bei 4°C oder 21°C 6, 12, 24, 36, 48, 72, 96 und 120 Stunden in der feuchten Kammer gelagert. Dann wurden Korneoskleralscheiben präpariert und diese 3 Tage bei 4°C in Optisol konserviert. Um einen etwaigen latenten Zellschaden auszuschließen, erfolgte anschließend eine zweitägige Organkultur bei 37°C in MEM-Medium mit 2% fötalen Kälberserum. Nach Färbung mit Trypanblau und Alizarinrot wurde der Befund fotodokumentiert und die endotheliale Zelldichte mit Hilfe computerunterstützter Morphometrie bestimmt. Die statistische Auswertung erfolgte durch eine Varianzanalyse. Verglichen mit der Endothelzelldichte eines Vergleichkollektivs frischer Schweinehornhäute (3733 ± 291 Zellen/mm2) fand sich in der bei 4°C vor Konservierung gelagerten Gruppe eine signifikante Abnahme erst nach 96stündiger Lagerung in der feuchten Kammer (p = 0,025). Hornhäute, die vor Konservierung bei 21°C in der feuchten Kammer aufbewahrt worden waren, wiesen nach 48 stündiger Lagerung den gleichen Befund auf (p < 0,001). Wir schließen aus diesen experimentellen Ergebnissen, daß die Empfehlung für die Kurzzeitkonservierung von Spenderhornhäuten nur Gewebe mit kurzen post-mortem Zeiten zu verwenden, große Mengen potentiell für den klinischen Einsatz verfügbaren Spendergewebes ausschließt.SummaryShort-term storage is a simple, effective technique for preservation of donor tissue for penetrating keratoplasty. It is recommended however, not to use tissue with post-mortem times exceeding six hours. The purpose or our study was to investigate whether this recommendation could be verified in an experimental study. Eye balls of freshly slaughtered pigs were stored for 6, 12, 24, 36, 48, 72, 96, and 120 hours at 4°C or 21°C in a moist chamber. After dissection of corneoscleral rims the tissue was stored for three days in Optisol at 4°C. Subsequently, corneas were stored in organ culture for two days at 37°C in order to exclude latent cell damage. Evaluation was performed by trypan blue-alizarin red staining. Corneal endothelial cell density was determined by computer assisted morphometry. Statistical calculation was performed by oneway analysis of variance. Corneas, that had been stored in a moist chamber at 4°C before short-term storage displayed a significant decrease in endothelial cell density (p = 0.025) after 96 hours compared to fresh tissue. Corneas stored at 21°C before short-term storage showed significantly reduced cell counts after 48 hours (p = 0.001). We conclude from these experiments the recommendation to use only tissue with short post-mortem times may exclude large amounts of potentially feasable corneas for penetrating keratoplasty.

Visusprognose nach perforierender Keratoplastik

Zusammenfassung160 Patienten wurden vor perforierender Keratoplastik und ein Jahr später untersucht. Als Parameter für die individuelle Zufriedenheit wurde der beste korrigierte Visus untersucht. Nach einem Jahr waren 87% der Transplantate klar geblieben. Die Visusverbesserung war bei Patienten verschiedener Diagnosegruppen ohne andere visusmindernde Erkrankungen vergleichbar. Wenn diese jedoch einbezogen wurden (34% der Patienten), kam es zu einer deutlichen Einschränkung der Visusprognose, die mit dem Durchschnittsalter für die jeweilige Diagnosegruppe korrelierte. Heutige Standardmethoden für die Lagerung von Spendergewebe, Operationstechniken und postoperative Behandlung haben eine so hohe Erfolgsquote, daß bei der Indikationsstellung das Vorhandensein anderer ophthalmologischer Erkrankungen das funktionelle Resultat mehr von diesen als von der Operation an sich abhängt. Der Patient sollte vor der Transplantation hierüber aufgeklärt werden, um für ihn entäuschende Ergebnisse zu vermeiden.SummaryVisual acuity of 160 patients was evaluated before and one year after penetrating keratoplasty in order to evaluate individual satisfaction. One year after transplantation 87% of the grafts had remained clear. Patients without other ophthalmologic diseases gained the highest increase in visual acuity. However, 34% of the patients suffered from other diseases, leading to a significant decrease in visual acuity one year after surgery. These findings were correlated to the mean age of established diagnosis groups. Due to current standards of donor tissue storage, surgery techniques, and treatment after surgery, penetrating keratoplasty can be considered to be highly successful. Thus, other ophthalmologic diseases, being present along with disorders leading to corneal transplantation, gain a high impact on prognosis for visual acuity and individual satisfaction, respectively.