P24-A114†Bringing together the eye banking community throughout Europe and beyond - promoting eye donation in Africa.

PURPOSE: It is estimated that globally there are more than 12.7 million corneal blinds with the vast majority of those living in the developing world. There is huge demand for corneal transplants worldwide as currently only one out of 70 patients can be provided with a cornea.Following the spirit of EEBA in bringing together the international eye banking community we present on our efforts and vision in contributing to the elimination of avoidable blindness in Africa by promoting sustainable eye donation programs. METHODS: At the congress of the South African Tissue Bank Association (SATiBA) in November 2022 a dedicated Round Table Discussion takes place on eye donation in Africa, organized by the World Union of Tissue Banking Associations (WUTBA) together with the Global Alliance of Eye Bank Associations (GAEBA), SATiBA and the German Society for Tissue Transplantation (DGFG). Individuals, national and global players in tissue medicine meet aiming to promote and advocate corneal donation in sub-Saharan Africa to establish patient care that is self-sustaining from within the countries.In preparation for the meeting a questionnaire was completed by the participants to understand the current situation in individual countries: Responses by ophthalmologists, tissue bankers, awareness and tissue donation coordinators from Kenya, Uganda, Nigeria, Ethiopia, and South Africa were evaluated. RESULTS: The survey revealed that all countries are establishing national health acts with references to tissue donation or have them in place with regulations still to be detailed. These are fundamental to strengthen confidence in tissue donation and to start developing donation infrastructures. In all countries there is doubt about donation after death showing the need for advocacy towards the public.The aim of the Round Table is creating a momentum of networking and sharing experience to support the African countries in building local infrastructures and becoming independent from tissue imports in the future. CONCLUSION: What frameworks must exist to successfully establish donation programs in Africa? What help can be provided by countries and organizations that have stable donation programs? These and other questions will be attempted at the Round Table. Bringing together experts, bundling synergies, and creating a momentum to promote cornea donation on social, political, and community level will be a step towards the vision of creating a world in which nobody is needlessly visually impaired.

P04-A115†Serum containing organ culture of the human cornea - still state of the art?

PURPOSE: For decades, human corneas are prepared and stored in specialized tissue banks prior to transplantation. Especially in Europe, storage takes place in 'organ culture', the storage in cell culture medium at approximately physiological temperature. Traditionally, a serum-containing medium is used for this purpose. However, the use of fetal calf serum has considerable disadvantages: there is a risk of disease transmission, availability may not always be guaranteed in the necessary quality, there are considerable differences from batch to batch, which is associated with batch testing required in each case, and last but not least, the extraction of serum from unborn calves is an ethical issue. METHODS: In recent years, several studies have focused on the improvement of organ culture conditions for donor corneas, including different serum-free media and alternative deswelling substances. Meanwhile, media are on the market which seem to be equivalent to serum-supplemented MEM. Nevertheless, serum-free medium has not yet found its way into routine organ culture of corneas. RESULTS: Our own preliminary studies have shown that despite the promising approaches, no satisfactory overall result could be achieved. Since only maintenance metabolism is required for storage of corneas until transplantation, in principle cultivation in the conventionally used medium seems possible without addition of serum at all. Corneas stored in this way had comparably endothelial cell density (ECD) to their counterpart stored in serum-supplemented medium. However, during the final evaluation after deswelling, the ECD dropped drastically.Engelmann et al. started research on the use of serum-free culture medium (SFM) for a long time and comparable or even superior ECD and viability could be demonstrated. So far, however, it has not been possible to define a deswelling medium adapted to these conditions.Also, a serum-free storage medium developed by Eurobio (CorneaSyn) could not completely convince, because although ECD of the examined corneas remained constant, the morphology of the cells changed. CONCLUSION: Since it is essential to intensify efforts towards a serum-free system it is planned to test serum substitutes and, if possible, also to replace the de-swelling additive dextran with a less harmful alternative to guarantee the quality of cornea grafts in the future.

8†The donor of tomorrow: challenges posed by the pandemic, demographic change, and increased transplant requirements.

In the Corona pandemic, the importance of donor health for the supply of patients with high-quality transplants has once again become particularly apparent in the field of cornea donation.And there are further challenges ahead: Due to new operation methods such as lamellar techniques an earlier stage of disease can be treated hence patients are being operated at younger ages. At the same time, with demographic change, potential donors are getting older.Therefore, the demand for a high-quality transplant without pre-operations seems to be difficult to fulfil in the future. This is particularly important in the highly developed industrialised countries, where the indications for corneal transplantation are different and the expected quality characteristics are therefore other than in emerging or developing countries, for example. At the same time, the new surgical methods present the tissue banks with new tasks to meet the surgeons' demands.In the DGFG network, the average age of corneal donors is currently 69.7 years while the requests for transplants with a high endothelial cell density (ECD) increase. The ECD continues to be one of the main criteria for a high-quality cornea and is more likely to be found in younger donors. As mentioned at the beginning, however, the average life expectancy in Germany is already currently around 80 years.It seems that it is impossible to find the perfect donor of tomorrow. With the increase in the need for high-quality transplants, the question must be asked whether donor shortage is a home-grown problem in industrialised countries. What developments need to be initiated to counter the trend towards donor shortage? Could greater flexibility at the medical and/or regulatory level be a solution? The presentation aims to shed light on these and other questions and would like to discuss this with the experts.

1†Crisis becomes the norm: how a non-profit network withstands the pandemic.

SARS-CoV-2 (corona virus) presents the world with new kinds of challenges. The crisis mode that persisted in many countries also put a strain on the German health system: on the one hand, through the treatment of patients infected with corona, and on the other hand through the cancellation and postponement of elective operations. This had a corresponding impact on tissue donation and transplantation. The effects of the pandemic-related restrictions can be reflected by the rate of corneal donation in the DGFG network: With the beginning of the first closure in Germany, donation and transplant numbers decreased by almost 25% from March to April 2020. After a recovery during summer, the activities were again restricted from October onwards due to increasing infection numbers. Subsequently in 2021 there was a similar trend.The already careful screening of potential tissue donors was expanded in accordance with the guidelines of the Paul-Ehrlich-Institute. However, this important measure led to an increase in discontinued donations due to medical contraindications from 44% in 2019 to 52% in 2020 and 55% in 2021 (Status Nov 2021). Nevertheless, the donation and transplantation result from 2019 was exceeded and DGFG was able to maintain patient care in Germany on stable level compared to other European countries. This positive result is partly due to an increased consent rate of 41% in 2020 and 42% in 2021 due to a higher sensitivity in the population to health issues during the pandemic. In 2021, the situation stabilised again, although the number of donations that could not be realised due to corona detection in the deceased continued to increase with the waves of infections that occurred.Low losses in donation and thus in the supply of transplants for patients seem to be due to the fact that a nationwide network such as the DGFG can respond flexibly to changing requirements. For example, if the number of COVID-19 infections varies between regions, it is possible to react to the local conditions to continue donation and processing where possible and allow allocation to regions where transplantation can take place.In summary it has been shown that efficient donation programs, resilient network structures, awareness of population for tissue donation and effective precautionary measures ensure a safe patient care with corneal transplants also in pandemic times.

11†Growing together in diversity - Indo-German cooperation enhancing eye donation in North India.

In India, the most densely populated state is Uttar Pradesh in the Northern region. This state has a huge base of corneal blind population due to cornea infections, ocular trauma, and (chemical) burns.Successful cornea transplantation using human post-mortem donated cornea is a treatment modality. In India lack of availability of donated cornea is a public health challenge. Thus, there is great need to reduce the huge demand and supply gap by increasing the donations for supply of cornea to patients.The Eye Bank at the Dr. Shroff's Charity Eye Hospital (SCEH) and the German Society for Tissue Transplantation (DGFG) collaborate in a project to enhance cornea donation and eye bank's infrastructure in Delhi. The project is supported by the Hospital Partnerships funding programme which is a joint initiative of Germany's Federal Ministry for Economic Cooperation and Development (BMZ) and the Else Kröner-Fresenius Foundation (EKFS) and carried out by the German Society for International Collaboration (GIZ GmbH).The project aims to increase the number of cornea donations by the SCEH eye bank through establishing two new eye collection centers where donation is coordinated and that are integrated into the existing and well-established eye bank and donation infrastructure of SCEH. Further, data management of the eye bank will be improved by developing a concept for an electronic database system that allows faster monitoring and evaluation of the processes. All activities are carried out according to a defined project plan. The basis of the project is an open-minded analysis and understanding of processes of both partners in relation to the respective legislations plus the environment and conditions in both countries.Aside from intercultural exchange and personal contacts both partners benefit from mutual on-site visits and exchanging best practices in eye donation and banking as well as sharing expertise in research topics.This project is a great example on how strong and sustainable relationships can be build across the globe improving the infrastructure for cornea donations to help corneal blind patients.

Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses.

Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting ß2-microglobulin (ß2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.

Cornea Procurement and Processing up to 72 Hours: No Risk for Cornea Transplant Quality.

BACKGROUND: The realization of tissue donations is bound to a tight timeframe. Depending on the type of tissue, time limits are specified within which the donation must be procured and processed. Otherwise, there is a risk of tissue quality loss with increasing time intervals from cardiovascular arrest. According to the European Directorate for the Quality of Medicines and HealthCare (EDQM) guide, cornea must be procured and processed within 72 h after death. The question arises whether this time interval has an influence on the quality of transplanted tissues and how it affects the accomplishment of tissue donations. METHODS: In order to obtain information on this, the numbers of tissue donations in the network of the German Society for Tissue Transplantation (DGFG) were evaluated as a function of the death to retrieval time (DRT) as well as the death to preservation time (DPT). For this purpose, 21,454 database entries of cornea donations made in the period from 2014 to 2018 were included. RESULTS: The results show that nearly 50% of donations realized in the DGFG network could be processed only 48 h or later after cardiovascular death due to the opt-in regulation in Germany. For these donations, there seems to be a higher discard rate compared to donations taken earlier. Nevertheless, there is a transplantation rate for these grafts of more than 65%, which is comparable to average transplantation rates stated in the literature. CONCLUSION: All corneas finally selected for transplantation must meet the specified quality parameters. Since this naturally also applies to transplants that could only be procured at later time points, it can be concluded that DPT up to 72 h for corneal tissue is adequate and has no influence on the quality of corneas that are ultimately transplanted.

Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.

PURPOSE: Subretinal transplantation of retinal (RPE) or iris (IPE) pigment epithelial cells has been advocated as a treatment for retinal degeneration. However, to our knowledge, in patients with age-related macular degeneration no significant beneficial effects on vision have been shown. Since the transplanted cells did not appear to maintain a healthy avascular and neuroprotective environment, we postulate that it will be necessary to transplant cells that express elevated levels of anti-angiogenic and neuroprotective activities. In our study, we provide a protocol for the efficient stable gene transfer and sustained gene expression of pigment epithelium-derived factor (PEDF), a potent anti-angiogenic and neuroprotective factor, using the nonviral Sleeping Beauty transposon system (SB100X). METHODS: Pigment epithelial cells were electroporated with a Venus reporter or a PEDF encoding plasmid, controlled by either CMV or CAGGS promoters. Transfection efficiencies and protein expression stability were evaluated by flow cytometry and immunoblotting. Gene expression profiles were analyzed by RT-PCR. RESULTS: SB100X-based delivery resulted in efficiencies of 100% with the Venus gene and 30% with the PEDF gene. Cell sorting enabled establishment of pure PEDF-transfected ARPE-19 populations. Transfected RPE and IPE cells have been shown to maintain stable PEDF secretion for more than 16 and 6 months, respectively. CONCLUSIONS: Transfection using the nonviral SB100X vector system avoids complications associated with viral gene delivery. SB100X-mediated transfer allows for stable PEDF gene integration into the cell's genome, ensuring continuous expression and secretion of PEDF. Stable expression of the therapeutic gene is critical for the development of cell-based gene addition therapies for retinal degenerative diseases.

Presence of xenogenic mouse RNA in RPE and IPE cells cultured on mitotically inhibited 3T3 fibroblasts.

PURPOSE: Mitotically inhibited 3T3 fibroblasts are used as feeder layers to culture a variety of cells. However, transplantation of human cells cultured on mitotically arrested mouse cells poses potential risks, such as disease transfer and contamination with 3T3 cells. Bovine RPE and IPE cells were cultured on mitomycin-treated 3T3 fibroblasts, to examine cell characteristics and contamination by 3T3 products. METHODS: IPE or RPE cells cultured on mitomycin-treated 3T3 fibroblasts were evaluated for adhesion, morphology, and tight junction formation by microscopy and immunohistochemistry. ROS phagocytosis was used to examine functional activity. Gene expression was evaluated by quantitative real-time PCR. RESULTS: In the presence of 3T3 fibroblasts, primary IPE and RPE cells adhere, spread and acquire a hexagonal shape within 12 hours. When cultured on 3T3 fibroblasts, IPE and RPE cells exhibited stable expression of pigment epithelial genes, but expression of mouse collagen type I was also observed. CONCLUSIONS: Culturing IPE and RPE cells on mitomycin-treated 3T3 fibroblasts resulted in rapid adhesion and growth of primary pigment cells. However, the presence of potentially hazardous xenogeneic mRNA of mouse origin in the cultures limits the use of these cells for transplantation.

Preservation of photoreceptors in dystrophic RCS rats following allo- and xenotransplantation of IPE cells.

PURPOSE: To examine whether iris pigment epithelial (IPE) cells transplanted into the subretinal space of Royal College of Surgeons (RCS) rats have the ability to rescue photoreceptors. METHODS: Rat IPE (rIPE) or human IPE (hIPE) cells were transplanted subretinally in 23-day-old RCS rats. Sham injection and transplantation of ARPE-19 cells served as controls. After 12 weeks, eyes were evaluated for photoreceptor survival by morphometric analysis and electron microscopy. RESULTS: Morphometric analysis showed photoreceptor rescue in all transplanted and sham-injected animals (number of photoreceptors/300 microm retina+/-sd: rIPE 41.67 +/- 28; hIPE 29.50 +/- 16; ARPE-19 36.12 +/- 21; sham 16.56 +/- 6) compared to age-matched, control rats (number of photoreceptors/300 microm retina+/-sd: 9.71 +/- 4). Photoreceptor rescue was prominent in IPE cell-transplanted rats and was significantly greater than sham-injected eyes (p = 0.02 for rIPE and p = 0.04 for hIPE). CONCLUSION: Since IPE cells transplanted into the subretinal space have the ability to rescue photoreceptors from degeneration in the RCS rat without any harmful effects, IPE cells may represent an ideal cell to genetically modify and thus carry essential genetic information for the repair of defects in the subretinal space.

The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes.

The transplantation of pigment epithelial cells as a therapeutic modality for retinal degeneration requires that the transplanted cells form a monolayer in the subretinal space that will establish communication with photoreceptors. Since previous studies have shown that transplanted cells in suspension do not form a monolayer, it will be necessary to transplant preformed pigment epithelial cell monolayers at the location of the exposed photoreceptors. To establish cell monolayers, retinal pigment epithelial (RPE) cells were cultured on ultrathin collagen membranes. Cells were examined for morphology, for characteristics of differentiation and viability. Membrane degradation and long-term biocompatibility in vivo were assessed following subconjunctival and subretinal implantation in rabbits. These studies have shown that RPE cells adhere, proliferate, form monolayers, and acquire differentiated properties on a collagen membrane that has features similar to Bruch's membrane. Membranes transplanted subconjunctivally and subretinally exhibit excellent biocompatibility without any evidence of inflammation or rejection. RPE cells cultured on collagen membranes acquire differentiated characteristics similar to those of RPE cells in vivo and form complete monolayers that are amenable to be transplanted to the subretinal space. The collagen membranes are non-toxic and do not elicit any rejection or inflammatory response when implanted subconjunctivally or subretinally in rabbits.

Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.

PURPOSE: The purpose of this study was to assess survival of adult mammalian neurosensory retina cultured in contact with the layer of a choroid-retinal pigment epithelium (RPE) explant. METHODS: The entire adult porcine neurosensory retina and RPE-choroid layer were placed in tissue culture by juxtaposing both tissues in their original orientation. Culture of the neurosensory retina alone and freshly prepared retina were used as control. After 3 days in culture retinal explants were fixed and processed for immunohistochemistry and TUNEL technique. RESULTS: We observed limited nuclei loss and significant reduction in apoptotic cells in nuclear cell layers (GCL, INL, and ONL) and decreased Muller cell hypertrophy in retina-RPE cultures compared to retinal cultures alone. In addition, cultures were characterized by reduced upregulation of GFAP, vimentin as well as S100 and increased glutamine synthetase expression. CONCLUSIONS: As any tissue culture model, retinal tissue culture is a short-term system and since degenerative processes begin quite early it may be a good model to investigate degenerative processes in the retina. However, our model of culture of retina adjacent to the RPE-choroid layer improves the maintenance of neural retina as evidenced by reduced apoptosis in nuclear cell layers (GCL, INL, and ONL) and reduced gliosis as indicated by the diminished expression of glial-specific proteins and increased glutamine synthetase compared to cultures of retina alone. Thus the retina-RPE-choroid culture system can enable the evaluation of interactions between RPE and neural retina, the role of signaling molecules as well the effect of pharmaceuticals on retinal biology.

Effects of bevacizumab (Avastin) on retinal cells in organotypic culture.

PURPOSE: Repetitive intravitreal injections of bevacizumab are a successful treatment option for exudative age-related macular degeneration (AMD). The aim of this study was to evaluate the toxicity of bevacizumab in the adult mammalian neurosensory retina in culture. METHODS: Adult porcine neurosensory retinas were cultured adjoined to the retinal pigment epithelium-choroid layer (retina-RPE-choroid complex) in static culture for 3 days, whereas neural retinas alone were cultured in a perfusion chamber for 3 days. Bevacizumab was added to the culture and perfusion medium at three concentrations (0.25 mg/mL [n = 6], 0.5 mg/mL [n = 6], and 1.25 mg/mL [n = 6]). Retina-RPE-choroid complex and neural retinas alone cultured without bevacizumab were used as controls. After 3 days in culture, the neural retinas alone and the retina-RPE-choroid complexes were analyzed histologically and immunohistochemically for the expression of glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase, rhodopsin, smooth muscle actin (SMA), and apoptosis. RESULTS: No toxic effects on ganglion or photoreceptor cells were observed at any concentration of bevacizumab. The expression of GFAP and vimentin was slightly increased in Müller cells, whereas glutamine synthetase and rhodopsin were unaffected by bevacizumab. However, significantly enhanced SMA expression in retina blood vessels was observed in retinas cultured in the presence of bevacizumab. CONCLUSIONS: Bevacizumab was well tolerated by ganglion and photoreceptor cells even at concentrations fivefold higher than those used clinically. The increased expression of SMA is an indication of the loss of functional VEGF modulating smooth muscle cells in mature vessels.