Jürgen Bednarz

Prospects for endothelial transplantation

BACKGROUND The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells (HCEC), transplantation of these cells may be an alternative therapeutic option. MATERIALS AND METHODS In this review methods for the in vitro cultivation of HCEC and their transplantation onto the Descemet membrane of donor corneas are described. RESULTS In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors. Dependent on the culture conditions, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behaviour. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation onto donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation status of the cells in vitro. Highest cell numbers up to 3000 cells/mm2 were achieved using a SV40-transformed HCEC-cell line. Monolayer integrity could be demonstrated by positive staining for integrins and light junction proteins, and pump function of the newly established endothelium was proven by perfusion studies. CONCLUSIONS Methods to transplant HCEC onto human denuded corneas have been successfully established to reconstruct human corneas. Recent developments in genetic manipulation of cells and tissue engineering will be of great help in constructing suitable corneas for keratoplasty. Thus corneal endothelial cell transplantation is one of the promising future possibilities to provide corneas of high quality for patients. Furthermore, improvement of the transplantation technique may lead to a method to directly manipulate the diseased endothelium of patients with corneal endothelial dystrophies.

DIFFERENT CHARACTERISTICS OF ENDOTHELIAL CELLS FROM CENTRAL AND PERIPHERAL HUMAN CORNEA IN PRIMARY CULTURE AND AFTER SUBCULTURE

SummarySeveral methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells.

BACKGROUND Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.Hintergrund: Das humane korneale Endothel ist in vivo nur eingeschränkt teilungsfähig und geschädigtes Endothel kann bisher nur durch die Transplantation einer Spenderhornhaut ersetzt werden. Die Transplantation von isolierten und in-vitro-kultivierten Endothelzellen könnte zukünftig eine therapeutische Alternative darstellen. Material und Methode: Beschrieben werden die Verfahren zur Kultivierung von humanen kornealen Endothelzellen und eine Methode zur Transplantation dieser Zellen auf die Descemetsche Membran von Spenderhäuten in vitro.Ergebnisse: In vitro kultivierte Endothelzellen wiesen kulturabhängige, aber auch unabhängige phänotypische Veränderungen und unterschiedliches Proliferationsverhalten auf. Untersuchungen auf molekularbiologischer Ebene zeigten, daß fibroblastische (dedifferenzierte) und endothelähnliche (differenzierte) Endothelzellen ein unterschiedliches Expressionsmuster für Rezeptoren von Wachstumsfaktoren aufwiesen. Weitere Untersuchungen deuteten darauf hin, daß Endothelzellen, die von der Randzone von Spenderhornhäuten isoliert wurden, eine stärkere Proliferationskapazität besitzen als zentral isolierte Zellen. Die Vermehrung der Endothelzellen in vitro ermöglichte die Transplantation der Zellen auf Spenderhornhäute in einem In-vitro-Modell. Nach Transplantation bildeten diese einen Monolayer aus, dessen Morphologie und Zelldichte von der Differenzierung der Zellen abhängt. Die DNS-Synthese war vorwiegend in Zellen der Hornhautperipherie feststellbar. Schlußfolgerung: Die Hornhautperipherie scheint eine regenerative Zone für das korneale Endothel darzustellen. Die auf der natürlichen Matrix (ECM) nach der Transplantation früh einsetzende Ausbildung eines Monolayers mit Kontaktinhibition weist auf zusätzliche inhibitorische Faktoren aus der Descemetschen Membran hin, welche die Proliferation der Zellen beeinflussen. Weitere Untersuchungen zur Regulation der Proliferation und Differenzierung humaner kornealer Endothelzellen in vitro und nach Zelltransplantation könnten dazu führen, in naher Zukunft ein selektives Verfahren zur Behandlung von kornealen Endothelzellverlusten zu etablieren.

Calcium influx induced by activation of receptor tyrosine kinases in SV40-transfected human corneal endothelial cells

This study was undertaken to investigate electrophysiological properties of immortalized SV40-transfected human corneal endothelial cells (HCEC-SV40) combined with the analysis of intracellular Ca(2+) responses mediated by ligands for receptor tyrosine kinases (RTK). In addition, the effects of several tyrosine kinase inhibitors were tested on Ca(2+) inflow mediated by induction of capacitative calcium entry (CCE). Patch-clamp techniques and measurements of the intracellular free Ca(2+) ([Ca(2+)](i)) by fura-2 were performed using HCEC-SV40. Stimulation of fibroblast growth factor receptors (FGFR) (e.g. by basic-FGF) (10 ng ml(-1)) elicited activation of Ca(2+) permeable channels and a subsequent increase of cytosolic free Ca(2+) in HCEC-SV40. This effect could be disrupted by the L-type Ca(2+) channel blocker nifedipine (5 microM). In addition, nifedipine significantly reduced the magnitude of CCE. Inhibition of protein tyrosine kinases (PTKs) by genistein, lavendustin A, or tyrphostin 51 (all 5 microM) also led to a reduction of CCE in HCEC-SV40. This study demonstrates for the first time that L-type Ca(2+) channel activity in HCEC-SV40 is linked to the activity of FGF receptor tyrosine kinases. These data regarding Ca(2+) inflow through Ca(2+) channels could be useful for investigation of culture and vitality conditions of HCEC.

Effect of perfluorodecalin on human retinal pigment epithelium and human corneal endothelium in vitro.

Abstract · Background:Perfluorocarbon liquids are useful intraoperative tools in complicated vitreoretinal surgery. They are usually removed at the end of the procedure, but small amounts may remain in the eye. Recently, contradictory results have been reported on the damage in association with residual perfluorocarbon liquids in the eye. This study examined the effects of perfluorodecalin on human retinal pigment epithelium and corneal endothelium in vitro. · Methods:Vitality and proliferative capacity of cell cultures were measured after incubation with perfluorodecalin. Vitality of cell cultures were measured using the Life-Dead assay. Cell proliferation was determined by measuring incorporation of 5-bromo-2’-deoxyuridine into cellular DNA. Furthermore, endothelium of organ-cultured human corneas was examined after incubation with perfluorodecalin by photodocumentation. · Results:Both cell types showed less extinctions in the Life-Dead assay after incubation with perfluorodecalin. After removing perfluorodecalin from the cultures, cells showed the same capacity of proliferation as the control cells. Compared to control corneas, perfluorodecalin induced a decrease in endothelial cell density. In four corneas, endothelial cell necrosis was observed. · Conclusion:Decreasing extinctions in the Life-Dead assay after incubation with perfluorodecalin can be interpreted as showing a decreasing amount of vital cells. Because cell proliferation showed no significant changes the results suggest that perfluorodecalin may not be directly toxic to cells in vitro. It may exert an indirect or mechanical effect on cell function by impeding the normal metabolic exchange between endothelium and medium. Based on these results perfluorodecalin should be completely removed after operation.

Thirty years of cornea cultivation: long‐term experience in a single eye bank

Purpose:  To evaluate donor demographics, trends in donor tissue procurement and tissue storage over a long period.

Verlauf der Endothelzelldichte nach perforierender Keratoplastik Einfluss von spender- und empfängerabhängigen Faktoren

ZusammenfassungHintergrund. Nach einer Hornhauttransplantation kommt es zu einem Endothelzellverlust. Retrospektiv wurde untersucht, ob Empfänger- und Spenderalter, Postmortemzeit, Organkulturdauer, präoperative Zelldichte sowie die Diagnose einen Einfluss auf die Endothelzelldichte 2 Jahre postoperativ haben. Methode.Über einen Zeitraum von 2 Jahren wurden 120 Patienten nach perforierender Keratoplastik nachuntersucht. Anhand der ermittelten 2-Jahres-Endothelzelldichten wurden 4 Gruppen unterteilt und hinsichtlich der oben genannten Parameter verglichen. Ergebnisse. Die geringste Endothelzelldichte 2 Jahre nach Keratoplastik korrelierte mit dem höchsten Empfänger- und Spenderalter. Die höchsten Zelldichten zeigten Transplantate von Patienten mit der Diagnose Keratokonus. Der Zustand nach Rekeratoplastik korrelierte dagegen mit einem vergleichbar hohen Endothelzellverlust im Vergleich zur Ausgangszelldichte (nicht signifikant). Schlussfolgerung. Die Daten weisen darauf hin, dass das Alter und wahrscheinlich auch die Diagnose den Endothelzellverlust postoperativ beeinflussen. Verlängerte Nachuntersuchungszeiten und größere Patientenzahlen können hier weiteren Aufschluss geben.AbstractPurpose. Endothelial cell loss can be observed after keratoplasty, therefore in a retrospective study we analysed whether there is a correlation between donor age, recipient age, time post-mortem, preoperative cell density, diagnosis of the recipient and post-operative endothelial cell density. Methods. The endothelial cell densities of 120 patients who had undergone penetrating keratoplasty were examined over a period of 2 years. We divided the patients into four groups based on the endothelial cell density over 2 years. We examined the groups with regard to the parameters given above. Results. The lowest postoperative cell densities 2 years after keratoplasty showed a high correlation with the highest donor and recipient age. Even the lowest preoperative cell density was found in this group. Patients who underwent keratoplasty because of keratoconus had the highest cell densities after 2 years and also the lowest donor and recipient age. The preoperative cell density was also highest in this group. Discussion. The results indicate a correlation between increasing donor and recipient age, decreasing preoperative cell density and loss of endothelial cells 2 years after penetrating keratoplasty. Patients with the diagnosis keratoconus should also receive transplants with higher donor age.

Recent developments in cornea transplantation (1997-1999).

Between 1997 and 1999 a steady increase in cornea donations was achieved, but the number of transplantations remained stable because many grafts did not pass quality control. Intermediate organ culture of entire bulbi was examined as a possible solution to reduce post-mortem times and increase suitability for transplantation.