P25-A123†Indo - German GIZ collaboration - impact and way forward in global eye banking.

PURPOSE: Geographical imbalance in cornea supply is a key feature of global eye banking. Most countries of South Asia particularly India suffer from donor cornea shortage which limits the number of keratoplasties, thereby aggravating the already high burden of removable blindness. The purpose of the project is to identify and cross-pollinate best practices from two leading eye banking institutions in India and Germany, and thereby improve service delivery of both systems. The project is supported by the GIZ Hospital Partnerships funding program on behalf of the Federal Ministry for Economic Cooperation and Development (BMZ) with a co-financing by the Else Kröner-Fresenius Foundation (EKFS). It started in 2021 and will last upto 2023. METHODS: A joint expert group from both organisations conducted a series of workshops to identify the areas of intervention and specific practices to be introduced at the Indian partner's region. The overall increase in cornea collections and transplants, documented systemic improvement measures and research output were defined as the key outcomes. RESULTS: Interim results are presented here. Two interventions identified were expansion of catchment area of cornea collection in India, and improved information management system to monitor the progress and efficiency of the collection centres. Under the former intervention, the hub-and-spoke model from the German partner was introduced to the most populous state of India through establishment of two new cornea collection centres (spokes) for Hospital based Cornea collections. In six months these centres have supplied 79 donor corneas leading to 63 transplants at the hub. Under the latter intervention, the specifications of a baseline data capture and operations management system which can be used in low resource settings are being developed. CONCLUSION: The initiative has shown how best practice from one geography can be adapted and successfully implemented in another geography , Furthermore, the public knowledge resources created in the project can be used by other eye banks to advance eye banking in their respective countries.

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.

New Safety Aspects in Corneal Donation-Studies on SARS-CoV-2-Positive Corneal Donors.

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT-PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT-PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT-PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT-PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

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.

Risk of SARS-CoV-2 virus transmission from donor corneal tissue: A review.

Since the outbreak of respiratory coronavirus disease (COVID-19) caused by the coronavirus SARS-CoV-2, there is an ongoing discussion about whether the virus could be transmitted through corneal transplantation from donor to recipient. The purpose of this review was to summarize the current knowledge in the scientific community to provide aid in risk evaluation for potential virus transfer by corneal transplants. Literature was searched in PubMed.gov for relevant articles on coronavirus in conjunction with cornea processing, cornea transplantation and eye banking. Further, guidelines of health authorities and eye banking associations were reviewed. Studies have shown that SARS-CoV-2 RNA can be detected in ocular swabs and/or fluid of patients with COVID-19. However, the risk of SARS-CoV-2 virus transmission through these ocular tissues or fluid of patients is judged differently. To date, per literature and official guidelines, no evidence of viable virus in ocular tissue and no cases of transmission of SARS-CoV-2 via tissue preparations have been reported.

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.

Human Amniotic Membrane: A review on tissue engineering, application, and storage.

Human amniotic membrane (hAM) has been employed as scaffolding material in a wide range of tissue engineering applications, especially as a skin dressing and as a graft for corneal treatment, due to the structure of the extracellular matrix and excellent biological properties that enhance both wound healing and tissue regeneration. This review highlights recent work and current knowledge on the application of native hAM, and/or production of hAM-based tissue-engineered products to create scaffolds mimicking the structure of the native membrane to enhance the hAM performance. Moreover, an overview is presented on the available (cryo) preservation techniques for storage of native hAM and tissue-engineered products that are necessary to maintain biological functions such as angiogenesis, anti-inflammation, antifibrotic and antibacterial activity.

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.

Comparison of preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) in a novel preloaded transport cartridge compared to conventional precut grafts.

To determine the safety and graft quality of eye bank precut and preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) after storage and shipping in a novel preloaded transport cartridge compared to precut grafts in a conventional viewing chamber. In this laboratory proof-of-concept study, 29 human donor corneas that were unsuitable for transplantation with a mean endothelial cell density of 1948 ± 260 cells/mm2 were prepared using liquid bubble technique for producing precut lamellar grafts. The grafts were either preloaded into novel transport cartridge (n = 16) or transferred into conventional Krolman viewing chamber (control, n = 13). Grafts were stored for 24 or 48 h in dextran-containing medium at room temperature and subjected to a shipping simulation. Endothelial cell loss (ECL) and morphology were determined at different steps. Endothelial cell viability staining was performed with calcein dye. Mean ECL in the preloaded transport cartridge was 0.7% ± 1.2% after 24 h and 3.4% ± 1.2% (p = 0.006) after 48 h storage and injection. In the control group the ECL was mean 1.6% ± 2.7% after 24 h compared to 3.7% ± 0.9% (p = 0.042) after 48 h. The slightly higher endothelial cell loss in the viewing chamber group after 48 h was not statistically significant compared to the preloaded transport cartridge (p = 0.8). Calcein staining was comparably low in all groups and correlated with the low ECL in both groups. DMEK grafts can be preloaded into a novel transport cartridge using a "no touch" technique, stored and shipped for up to 2 days in dextran-containing medium without significant ECL.

Precut DMEK Using Dextran-Containing Storage Medium Is Equivalent to Conventional DMEK: A Prospective Pilot Study.

PURPOSE: To compare the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK) either as precut or conventional Descemet membrane graft preparation under standard European eye bank organ culture conditions. METHODS: This was a prospective pilot study of patients receiving either precut or conventional DMEK. Graft preparation was performed using the liquid bubble technique. Precut grafts (n = 22) were prepared 1 day before surgery in the eye bank and stored in dextran-containing organ culture medium within a transport viewing chamber. Conventional grafts (n = 29) were prepared directly before surgery. End point criteria included the endothelial cell count (ECC), central corneal thickness, graft rejection rate, rebubbling rate, and best-corrected visual acuity after 1, 3, and 6 months. RESULTS: A post hoc matched analysis revealed no statistically significant differences between the 2 groups. The ECC in the precut and conventional groups was comparable with an EC loss of 34% and 35%, respectively, after 6 months. The early graft failure rate, best-corrected visual acuity, and central corneal thickness were comparable between the 2 groups. CONCLUSIONS: This pilot study shows a comparable clinical outcome after DMEK surgery for precut Descemet membrane grafts versus conventionally prepared grafts, using the liquid bubble preparation technique and storage conditions with dextran-containing medium.

miR-145 Is a Promising Therapeutic Target to Prevent Cornea Scarring.

Corneal scarring is an expected outcome of corneal injury or infection and is one of the major causes for visual loss. The formation of light-scattering myofibroblasts is thought to be the underlying cause of corneal haze formation. Recently, microRNA (miRNA) gene therapies have been proposed as novel approach for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in corneal myofibroblast differentiation and function. Analysis of human corneal scar tissue and transforming growth factor (TGF)-ß1-induced corneal myofibroblasts showed a 13- and 4-fold increase of miR-145, respectively, compared with healthy cornea and nonstimulated fibroblasts (p<0.01). Furthermore, myofibroblasts showed an increase in α-smooth muscle actin (α-SMA) expression and a decreased expression of Kruppel-like factor 4 (KLF4). These results indicated that TGF-ß1 increases miR-145 expression, which indirectly induces α-SMA expression via downregulation of KLF4, a known negative regulator of α-SMA. Consistently, miR-145 silencing in corneal myofibroblasts using a specific antimiR resulted in increased KLF4 and strongly decreased α-SMA expression. In addition, miR-145 inhibition also significantly decreased myofibroblast contractility, migratory capacity, and TGF-ß1 secretion, which are all thought to contribute to corneal scarring. Hence, miR-145 plays an important role in TGF-ß1-stimulated corneal myofibroblast differentiation and activation, which can be reversed by miR-145 silencing. Therefore, we suggest miR-145 as a promising therapeutic target for miRNA-based gene therapy to prevent or treat visual loss caused by corneal fibrosis.