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This study helps to estimate the contamination risk of a cultured cornea based on specific donor factors. However, donors with risk factors should not be generally excluded from cornea donation. Further studies including antibiograms might clarify whether a change in the antibiotic composition of the culture medium would be useful to deal with the increasing number of multi-resistant microbes.

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

PurposeRecombinant adeno‐associated viruses 2 (rAAV2) represent a nonpathogenic and safe alternative to other viral delivery systems. However, their transduction efficiency in corneal endothelial cells (CEC) is limited. As the level of transgene expression is dependent on the conversion of single‐stranded (ss)‐ into double‐stranded (ds)‐DNA, self‐complementary (sc)‐AAV vectors have been developed to circumvent this problem. The aim of this study was to evaluate the use of scAAV2 in terms of transduction efficiency in CEC. Additionally, the impact of transduction on cell viability was investigated.MethodsA human corneal endothelial cell line (HCEC‐12) as well as organ‐cultured human donor corneas were transduced with different titers of ss‐ or sc‐AAV2. Transduction efficiencies were compared by means of GFP‐transgene expression. GFP‐expression in HCEC‐12 cells was evaluated by flow cytometry over a period of 28 days. GFP‐expression in human donor corneas was analyzed by confocal microscopy on day 6. 7‐AAD staining and flow cytometry as well as MTT‐assay were performed to determine cell viability after transduction.ResultsGFP‐expression was significantly higher in cells transduced with scAAV2 than in cells transduced with ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. The highest transgene expression rate using scAAV2 was 86.9% compared to 80.5 % using ssAAV2. In human donor corneas GFP‐expression was observed in 72.2% (scAAV) and 44.1% (ssAAV) of CEC respectively. There was no significant difference between viability of transduced and untreated cells.ConclusionsScAAV2 vectors are an effective tool to enhance transduction efficiency in CEC. Allowing higher transduction rates with lower vector titers, this could improve AAV2‐mediated gene therapy to protect CEC in corneal allografts.

Purpose Graft rejection is one of the major causes of corneal transplant failure after keratoplasty, especially in case of an ocular or systemic inflammatory disease. To reduce the patient’s immune reaction against the corneal allograft, topical or systemic immunosuppression is a clinical necessity. This talk provides an overview of the benefits of systemic immunosuppression and of its indications. Methods The advantages and disadvantages of the use of systemic immunomodulatory drugs in the prevention of corneal allograft rejection after keratoplasty will be analyzed and demonstrated. Results Various systemic immunomodulatory drugs are available to prevent or to reduce the risk of corneal allograft rejection. The use of systemic immunosuppressive drugs may be limited by side effects. Conclusion Systemic immunosuppression prolongs corneal allograft survival and reduces the risk of graft rejections. While the use of systemic immunosuppressive drugs is beneficial in the perioperative stage, the long‐term‐use is restricted to high‐risk settings.

Purpose In the majority of European countries there is a shortage of donor corneas for corneal transplantations. In 2007, the EU Directive 2006/17/EC on ‘technical requirements for the donation, procurement and testing of human tissues and cells’ was introduced restricting the usability of post‐mortem donor blood sampling from 72 hours to 24 hours. As a consequence, post‐mortem time for donor cornea acquisition was shortened, resulting in a further reduction of corneal allografts. This study analyzed the effect of donor cornea post mortem‐time on medium contamination in organ culture storage. Methods Contamination rates during corneal organ culture during extended (2008‐9, Group I) versus restricted post‐mortem time protocols (2010‐11, Group II) were retrospectively analyzed. Data were collected at LIONS Cornea Bank North Rhine‐Westphalia, University Eye Hospital Duesseldorf, Germany. Results In the years 2008 and 2009 (Group I) 1272 corneal grafts with a post‐mortem time of 30.05 ± 15.77 hours were collected and cultivated in LIONS Cornea Bank NRW. After introduction of the new guidelines, the overall number of acquired donor corneas dropped by 29.88% to 892. Post‐mortem time was significantly lower during the restricted post‐mortem time protocols (24.2 ± 12.37 hours, p<0.05, t‐test). Interestingly, there was no significant difference of culture medium contamination rates between the two groups (Group I: 9,0%, n=115 versus Group II: 7.3%, n=65; Chi‐squared test). Conclusion Restriction of donor cornea acquisition time down to 24 hours seems to be without benefit regarding the contamination rates during corneal organ culture.

This talk gives an overview of the actual data about the correction of infant aphakia with rigid gas‐permeable contact lenses. The advantages and disadvantages of the use of rigid gas‐permeable contact lenses after congenital cataract surgery will be discussed. Also, we will present our own data regarding the visual outcome of aphakic children treated with rigid gas‐permeable contact lenses.

This talk gives an overview of different types of contact lenses and their present applications and future perspectives. The advantages and disadvantages of contact lenses will be discussed. Furthermore, we will present our own data regarding correction of infant aphakia and irregular astigmatism following PKP with rigid gaspermeable contact lenses and the use of soft silicone contact lenses as a drug delivery tool.