Objective: This validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC^TM blood culture bottles.

Methods and analysis: 10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium.
In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles.
In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTEC^TM vials (positive control). All samples were incubated in the automated BACTEC^TM blood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liquid chromatography.

Results: After 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated.
In group A , all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls.
In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis.

Conclusion: The use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTEC^TM Plus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

Purpose: To test the feasibility of a cell therapy approach to treat corneal endothelial (CE) disorders using an in vitro model of human corneal decompensation.

Methods: A CE decompensation model was established by removal of the Descemet membrane/endothelium complex from cadaveric human corneas in an air interface organ culture system (group 2) and compared with normal corneas (group 1). The posterior stroma of decompensated corneas was seeded with immortalized human corneal endothelial cells (HCEC-12) in group 3 and passage 0 primary human CE cells in group 4 corneas. Functional effects on stromal thickness were determined with histological analysis 3 to 10 days after cell therapy treatment.

Results: Removal of the Descemet membrane/endothelium complex in group 2 corneas resulted in a stromal thickness of 903 ± 86 μm at 12 hours compared with 557 ± 72 μm in group 1 corneas. Stromal thickness reduced from 1218 ± 153 μm to 458 ± 90 μm (63% ± 6%, P = 0.001) after cell transplantation in group 3 and from 1100 ± 86 μm to 489 ± 94 μm (55% ± 7%, P = 0.00004) in group 4. Posttransplantation histology demonstrated formation of a monolayer of corneal endothelium attached to the posterior stromal surface.

Conclusions: Direct transplantation of cultured human CE cells and immortalized HCEC-12 to bare posterior corneal stroma resulted in formation of anendothelial monolayer and restoration of stromal hydration to physiological thickness, demonstrating the feasibility of cell therapy in treatment of CE decompensation in a human in vitro model.

Summary: A spanish study by electronical microscopy on corneas for DMEK, stored in Tissue-C at 31°C, shows that there are morphological and structural differences between the center and the periphery of the grafted tissue.

Objectives: To evaluate the efficacy of Eusol-C as a corneal storage medium on the survival of donor endothelium.

Materials and Methods: Twenty-seven corneas not suitable for transplant were included in this study. All donor corneas were stored in Eusol-C at 4°C. Daily donor corneal endothelial cell counting was performed with an eye bank specular microscope. All corneas were discarded after the study process.

Results: Mean donor age was 51.3 ± 18.8 years (range, 25-94 y). The mean duration between death and corneal excision was 9.5 ± 6.7 hours (range, 3-23 h). Mean endothelial cell density was 2195 ± 383 cells/mm² at the beginning of the preservation (range, 1361-2899 cells/mm²). Donor endothelial cell density was between 1500 to 2000 cells/mm² in 9 corneas, 2000 to 2500 in 11 corneas, 2500 to 3000 in 5, and higher than 3000 in 2 corneas at baseline. Mean endothelial cell density was found 1658 cells/mm² on the eighth day of storage, with a mean endothelial cell loss rate of 24.5%. Corneas stored 9 to 24 days in Eusol-C had a rate of endothelial cell damage of 3.1% per day.

Conclusions: Although our results revealed a higher endothelial cell loss than previous reports, overall performance of Eusol-C in preserving the donor endothelium may be satisfactory for clinical use.

Objectives: To analyse 1.5-year data of our newly established eye bank and to evaluate the factors affecting donor quality.

Methods: Our bank’s donor cornea data between July 2013 and November 2014 were retrospectively analysed. The effects of donor age, sex, and time from death to harvesting on the findings of specular microscopy were assessed.

Results: A total of 139 corneas retrieved from 70 donors. The mean age of donors was 34.2±14.6 (5-64) years. The mean time from death to harvesting was 6.7±2.9 (1-13) hours; the mean time
from collection to transplantation was 5.2±2.8 (1-14) days. Age had a significant negative correlation with mean endothelial cell count (ECC), a significant positive correlation between mean cell area (MCA) and standard deviation (SD). Time from death to harvesting had a significant negative correlation with cell count and 6A; it had a significant positive correlation with SD, the coefficient of variation, and MCA.

Conclusion: According to the results of the present study, ECC, MCA, and SD levels were greater in younger donors. Endothelial
morphology was altered as the time from death to harvesting was prolonged; however, the alteration in cell morphology was not severe enough to alter transplantation success with the corneas being harvested within the first 13 hours.

Abstract: This study was conducted to compare the quantitative and qualitative indices of the donated corneas maintained in either Optisol-GS or Eusol-C storage media. In an ante-grade single blind study, two corneas from each donor with a death to preservation time of less than 30 h and with a minimum of “an apparent good cornea rating” were maintained in corneal storage media; randomly one in Optisol-GS and the other in Eusol-C. Slit-lamp biomicroscopic and specular microscopic examinations were performed on days 1 and 7. The results of the qualitative and quantitative indices and the final cornea rating were recorded. Statistical analyses were performed to evaluate any differences between the two media. 180 corneas from 90 donors with an age range of 29.3 ± 22.4 years were allocated in two groups: 90 corneas in Optisol-GS and the other 90 in Eusol-C. Five corneas in Optisol-GS and four corneas in Eusol-C were excluded from the study due to lack of specular images. There was no significant change of endothelial rating from day 1 to day 7 between two media (P = 0.175). As the maintenance time of the donated corneas increased, no significant difference was noted between the two groups in terms of endothelial indices, stromal edema, and Descemet’s folding. This study shows no superiority between Eusol-C and Optisol-GS in short term preservation of donated corneas.

Abstract: It has been recently shown that cold storage at 2-8°C is not neutral towards metabolic properties of the cornea. The aims of this study were to reveal: the kinetics of biochemical changes in human corneas during cold storage and the time of storage when the preserved corneas show the biggest biochemical similarities to in vivo ones. Twenty human corneas were obtained post-mortem from ten 60 to 71-year-old donors and preserved in Eusol-C at +4°C for 14 days. The control samples were the tissues stored at -80°C immediately after collecting them from cadaver eye globes. The daily changes in metabolic profiles of the samples were investigated with HR MAS 1H NMR spectroscopy. 25 metabolites were detected and assigned in the corneas. The significant differences in metabolic profiles between Eusol-C-preserved and non-preserved tissues, and between the corneas stored for 14 days comparing to 7-days samples, were revealed. The main differences between the samples were related to the levels of ATP, formate, lactate, glycine and butyrate in the tissues. The corneas preserved for 2 weeks were shown to have the biochemical contents more resembling the in vivo ones than the corneas preserved for one week. The results of this study suggest that from the biochemical point of view the cornea transplantation or any other procedure associated with a removal of the cornea from the storage medium should be performed after two weeks of the hypothermic preservation in Eusol-C.