Corneal Storage Medium Research Articles

Investigating the Influence of Temperature and Supplementation Timing on Antifungal Efficacy in Storage Medium for Corneal Transplantation.

Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue. Corneoscleral rims contaminated with Candida albicans (C.albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericinB or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2-8°C versus 2h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period. Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C.albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day2 or day6 showed a 99% or greater reduction of C.albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture. The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C.albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day2 or day6 was necessary during a 2-week preservation period.

Risk Factors of Corneal Donor Contamination and Post-Keratoplasty Infectious Complication

Introduction & Objectives : Infectious keratitis and endophthalmitis are rare yet devastating complications of corneal transplantation. Some studies have demonstrated increased incidence of infectious post-keratoplasty complications are related to contaminated corneal donor, although its risk factors remain controversial. This study provides concise review on possible risk factors of corneal donor contamination which related to post-keratoplasty infectious complications.
 Methods : This literature review was conducted by searching PubMed and Proquest for “corneal donor contamination” AND (“infectious complication” OR infection OR endophthalmitis) from the period of 2018-2023. Relevant articles were reviewed and selected for further understanding regarding various risk factors of corneal donor contamination leading to post-keratoplasty infectious complications and its preventive strategies.
 Results : The rate of corneal contamination varied between studies, ranging from 1,3 to 7,8%. The most common risks of contamination are donor’s diseases (e.g. cardiovascular, cerebrovascular accident) and prolonged time between death and donor retrieval. Longer preservation time (>5 days), environmental situation (temperature and humidity), and prolonged treatment duration on intensive care unit (>4 days) are also considered as its risk factors. Corneoscleral rim culture, addition of antifungal and antibacterial to corneal storage medium, and shortening of corneal processing time should be considered as probable preventive strategies.
 Conclusion : Donor diseases, prolonged donor retrieval time from death, and longer donor preservation and hospitalization duration, are associated with higher rates of corneal donor contamination. Rapid retrieval and donor handling time, together with corneoscleral rims culture, at least for fungus regarding its better predictive values, should be taken into consideration by corneal donors providing eye banks.

Assessment of performance and safety of Corneal Chamber hypothermic storage medium and PSS-L corneal rinsing solution in human and porcine corneas

PurposeTo prove the safety and performance of the hypothermic corneal storage medium "Corneal Chamber" and the rinsing solution "PSS-L" in support of the new Conformité Européenne (CE) certification process in...

Comparison of corneal endothelial cell density and morphology with Optisol GS and Life4C storage media in the eye bank: a 5-year retrospective analysis.

Optisol GS and Life4C are corneal storage media used by eye banks worldwide. We sought to determine if either solution was associated with superior corneal endothelial cell density (ECD) or morphology in a large cohort of donor corneas. From January 2016 through December 2020, 10,316 corneas from 5,624 unique donors were acquired and analyzed at Rocky Mountain Lions Eye Bank. In April 2019, Life4C replaced Optisol GS as the sole storage medium. We compared ECD and morphology before and after April 2019, and excluded corneas processed within the transition period. Univariable and multivariable regression analyses accounted for age, sex, tobacco use, heavy alcohol use, and diabetes. Only right corneas were analyzed to account for the correlation between eyes. Of 5042 right corneas analyzed, 3486 were stored in Optisol GS and 1556 in Life4C. There was no significant difference in ECD across groups (2794 vs. 2793 cells/mm2 in Optisol GS and Life4C, p=0.88). In multivariate analyses, there was no significant difference in corneal ECD (0.6 cells/mm2 higher with Life4C, p=0.96) or hexagonality (0.22% higher with Life4C, p=0.31). However, the coefficient of variation was significantly lower in the Life4C group (-0.0039, p=0.03). After adjustment for above factors, corneas in Life4C demonstrated a 3.1% decreased likelihood of exhibiting CV values greater than 0.40 (p=0.009). This study demonstrates comparable and favorable outcomes using both storage media and confirms their overall efficacy. The decreased CV in Life4C is not of clinically significant magnitude, but merits further research in clinical and long-term settings.

Anti-fungal efficacy of combination of amphotericin B with colistin and gentamicin in McCarey-Kaufman corneal preservation medium.

To curtail the potential of donor corneal tissue disseminating fungi to the recipient's eye, we evaluated the addition of amphotericin B to McCarey-Kaufman (M-K)-corneal storage medium supplemented with colistin. Amphotericin B was examined for its ability to inhibit the growth of Candida albicans and Aspergillus flavus using a microbroth dilution test and checkerboard assay in combination with only gentamicin and a combination of colistin, gentamicin, and amphotericin B. The safety on epithelium and endothelium was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimal inhibitory concentration of gentamicin was found to be >256 μg/ml against both C. albicans and A. flavus, whereas that of amphotericin B was found to be in a range of 0.25-0.5 and 1-2 μg/ml for C. albicans and A. flavus, respectively. According to the checkerboard assay, 80% (4/5) of C. albicans isolates and 100% (5/5) of A. flavus isolates responded synergistically to the combination of amphotericin B and gentamicin, but only 20% (1/5) of C. albicans isolates showed an additive effect. None of the tested isolates displayed antagonism. The combined effect of the three drugs also did not display any antagonistic effect. Additionally, the MTT assay reveals no toxic effect of the antimicrobials used on corneal epithelial and endothelial cells. In vitro experiments demonstrate that amphotericin B is not toxic to either epithelium or endothelium and is a promising additive to the M-K medium supplemented with colistin.

An experimental study to evaluate the effect of polymixin E (Colistin) alone or in combination with gentamicin in McCarey-Kaufman corneal preservation medium on various drug resistant bacterial and fungal isolates.

To assess the efficacy of the addition of polymyxin E (colistin) in the McCarey-Kaufman (MK) corneal storage solution against multi-drug resistant strains of Enterobacteriaceae, Staphylococcus aureus, and Candida spp. A standard micro broth dilution test and a checkerboard assay were performed for five multi-drug resistant (MDR) clinical strains of P. aeruginosa and five clinical strains of methicillin-resistant S. aureus (MRSA) and C. albicans against colistin and gentamicin alone and in combination. The minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI) were calculated to assess the efficacy of each combination. The MIC of colistin was in the range of 1-2 μg/mL for P. aeruginosa, whereas it was 256-1024 μg/mL against S. aureus. In comparison, the MIC of gentamicin was found to be 0.5-512 μg/mL and 0.5-8 μg/mL against P. aeruginosa and S. aureus, respectively. All five isolates of C. albicans did not exhibit any susceptibility to either colistin or gentamicin even at a concentration of ≥ 512 μg/mL each. The checkerboard assay was performed to evaluate the nature of the interaction of the combination of colistin and gentamicin. Based on the FICI, it was observed that the colistin and gentamicin combination has a maximum synergistic effect (FIC <0.5) in 80% (4/5) for S. aureus isolates, whereas the maximum additive effect (FIC >0.5-4) was 100% (5/5) for P. aeruginosa and the minimum additive effect was 20% (1/5) for S. aureus isolates. Antagonism (FIC ≥ 4) was not observed in any combination between the strains used in the study. Both colistin and gentamicin alone or in combination were, however, ineffective against Candida spp. The addition of colistin has an inhibitory effect on bacterial contamination that could be possibly caused by MDR strains and could potentially be considered as an additional additive in corneal storage media.

Temperature effects on the disappearance and reappearance of corneal-endothelium primary cilia.

To elucidate the specific functions of the primary cilia in corneal endothelial cells (CECs) by investigating the histological changes of corneal endothelium exposed at low temperature. Experimental study. This study involved corneas freshly obtained from Japanese white rabbits preserved in Optisol™-GS (Bausch & Lomb) corneal storage medium at 4°C for 0, 1, and 7days. Corneas preserved for 7days were also incubated at 37°C in culture media for an additional 2days. A rabbit CEC line was also preserved in Optisol™-GS at 4°C for 0 and 1day. The corneal endothelium specimens and CECs were then assessed by immunostaining and scanning electron-microscopy (SEM). Immediately post isolation, the CECs of the specimens showed positive immunostaining for primary cilia (i.e., approximately 20%) via anti-acetylated alpha Tubulin antibody and SEM observation. Primary cilia were found to have attenuated/disappeared on the corneal endothelium specimens preserved for 1 or 7days at 4°C. After an additional 2-day incubation at 37°C, primary cilia reappeared on the corneal endothelium specimens (approximately 20%). The disappearance of cilia during the preservation period was also observed in the immortalized CECs. The findings in this study using rabbit corneas indicate that the primary cilia of corneal endothelium preserved at low temperature disappeared, then reappeared after returning to body temperature, suggesting that temperature has a direct effect on the primary cilia of corneal endothelium.

Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation

ObjectiveTo study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with...

Effect of Descemet Membrane Endothelial Keratoplasty Graft Storage Time on Graft Elasticity.

The purpose of this study was to evaluate the effect of Descemet membrane endothelial keratoplasty (DMEK) graft storage time on its elastic properties, measured using atomic force microscopy (AFM). Twenty human corneas (from 10 donors), unsuitable for transplantation, were obtained from the eye bank (S. Fyodorov Eye Microsurgery State Institution, Moscow). Ten DMEK grafts were prepared and stored in the corneal storage medium, Optisol-GS at 4°C after preparation, and AFM analysis was performed within 12 hours after preparation (group A). Ten paired corneas from the respective donors were stored in Optisol-GS at 4°C for 1 week after preparation before AFM analysis (group B). Data were analyzed using the Hertz model for the evaluation of the Young modulus of elasticity. Force-distance curve analysis showed an increase in the Young modulus of elasticity in group B in comparison with that in group A, and the mean values were 10.4 ± 1.8 kPa and 6.77 ± 2.25 kPa, respectively (P < 0.001). There was no correlation between the Young modulus of elasticity and donor age (r = 0.110, P = 0.644), endothelial cell count (r = -0.145, P = 0.541), and procurement interval (r = 0.14, P = 0.755). A longer graft storage time in cold storage medium was found to significantly reduce the elasticity of the DMEK graft. Clinically, this could potentially influence the unfolding of the DMEK graft within the anterior chamber during surgery and the postoperative detachment rate.

Переживаемость кератоцитов и эндотелиальных клеток заднего послойного трансплантата роговицы, культивированных в модифицированной консервационной среде

Purpose. To study the survival of keratocytes and endothelial cells of a human donor cornea storage in the standard and the new media which was specifically designed for optimized cornea hydration. Material and methods. 2D cell cultures of keratocytes and endothelial cells obtained from the Eye tissue bank were used for culture in improved storage media over a period of 14 and 7 days subsequently. To confirm phenotype characteristics, the cells were stained by the following markers: for keratocytes – Lumikan, Keratocan, and α-smooth muscle actin; for endothelial cells – ZO-1 and Na/K-ATPase. The onset of apoptosis in cell culture of keratocytes were detected with Cytochrome C, BAX, and Caspase 3 and 8. Viability of cell cultures after the cultivation was carried out using a commercial set of "Live and Dead". Morphology of the endothelial cells was assessed using an electron scanning microscope. Results. It was shown that the 2D keratocyte culture cultured in the improved storage media expressed specific markers: Lumican, Keratocan, and did not express α-smooth muscle actin. There were no markers of apoptosis in the cell culture of keratocytes after 14 days of cultivation. Corneal endothelium cultured in the improved storage media expresses ZO-1, Na/K-ATPase and presented hexagonal cell shape morphology according to electron microscopy. Conclusion. The improved storage media allow to preserve the unique phenotype of keratocytes, with a slight decrease in proliferative cells activity during 14 days. The media maintain a viable and functional corneal endothelium for at least seven days of cultivation. Key words: cell culture; corneal endothelium; keratocyte; posterior lamellar graft, corneal storage media.

Solubilized ubiquinol for preserving corneal function

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol – the reduced and active form of coenzyme Q10 and a powerful antioxidant – significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or β-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

Study of the antiviral activity of the liquid corneal storage medium in relation to the herpes simplex virus in vitro

The study of antiviral activity of 7 samples of liquid corneal storage medium on a model of herpesvirus infection caused by the herpes simplex virus type 1 in Vero-cell using virological and statistical research methods was carried out. All the studied images of the corneal storage medium, including the Borzenka-Moroz base medium, did not have a cytotoxic effect on Vero cell culture. Out of of 7 samples of liquid corneal storage medium, 4 samples had reliable antiviral activity against HSV-1 when used under the therapeutic regimen (1 hour after infection) and under the preventive regimen (2 hours before infection). Antiviral activity was established in 2 samples containing the interferon inducer cycloferon at a concentration of 10 mg/kg and 30 mg/kg (sample 2, 3), in a sample containing the interferon inducer gamapren 15 mg/kg (sample 5), and in a sample containing a combination of drugs - 10 mg/kg cycloferon and an acyclic nucleoside analog-acyclovir 10 mg/kg (sample 6). According to the results of 2 test regimens, the maximum statistically significant inhibitory effect in relation to HSV-1 was detected in sample 6, containing a combination of drugs. Against the background of sample 6, the infectious activity of the test virus decreased by an average of 3.2 lg, the inhibition coefficient was 54.5%. The results of the study indicate the prospects of using types of media with antiviral activity (samples 2, 3, 5, 6) for storing donor corneas in order to increase the effectiveness of keratoplasty in patients with ophthalmic herpes.

Antimycotic Efficacy and Safety of a New Cold Corneal Storage Medium by Time-Kill and Toxicity Studies.

To evaluate a new corneal cold storage medium including an antimycotic tablet (Kerasave, AL.CHI.MI.A. S.r.l.). Kerasave and tryptone soy broth (control) were inoculated with 10 and 10 colony-forming units (CFU)/mL of 6 Candida isolates (Candida albicans [n = 4], Candida tropicalis [n = 1], and Candida glabrata [n = 1]). Minimum inhibitory concentrations (MICs) were determined using amphotericin B Etest strips. Sterile porcine corneas contaminated with 10 CFU/mL of each isolate were incubated in Kerasave and control at 4°C. Growth rate and Log10 reduction at 4°C at different time intervals were determined for liquid samples and tissue homogenates. Kerasave biocompatibility was assessed according to ISO 10993-5 and ISO 10993-10. No C. albicans or C. tropicalis colonies were recovered from Kerasave inoculated with 10 CFU/mL after incubation for 3 days at 4°C. C. glabrata was inhibited but not killed after 3 days at 4°C. Four of the 6 strains contaminated with 10 CFU/mL demonstrated a significant ≥ 3 Log10 reduction in media and tissue homogenates within 5 days as compared to controls (p < 0.01). Amphotericin B MICs ranged from 0.19 to 0.38 μg/mL for C. albicans (n = 3) and C. tropicalis (n = 1). C. glabrata showed reduced susceptibility (0.5 μg/mL) and 1 C. albicans was resistant to amphotericin B (≥ 1 μg/mL). Kerasave was not cytotoxic, irritating, or sensitizing according to the ISO standards. Kerasave showed high antifungal efficacy against susceptible fungal strains at 4°C in the presence and absence of corneal tissue. Resistant strains to amphotericin B were not eliminated by Kerasave. Kerasave is not cytotoxic, irritating, or sensitizing.

A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia.

The purpose of this study was to validate the sterility test of corneal culture and deswelling/transport media using a device for removal of antimicrobials before incubation in BACTEC™ automated system in two Italian Eye Banks. Corneal culture medium, TISSUE-C, and deswelling/transport medium, CARRY-C, were inoculated with 10-100cfu of six European Pharmacopoeia (EP) reference strains and either treated with medical device RESEP for removal of antimicrobials (RESEP+ group) or left untreated (RESEP- group) before injection into the BACTEC Plus bottles. The same steps were repeated in the absence of inocula with tryptone soy broth samples as negative controls, and the inocula were also directly injected in the BACTEC™ bottles as growth controls. All the samples were incubated in BACTEC™ automated system for7days, and the time to detection of microbial growth was recorded automatically. At both the Eye Banks, in the RESEP+ groups, microbial growth was detected in 100% of samples. In the RESEP- group, the method sensitivity ranged from 66.7 ± 21.1 to 88.9 ± 6.4% for TISSUE-C samples while for CARRY-C samples the method sensitivity ranged from 94.5 ± 5.1 to 100%. The method specificity corresponded to 100% for all the groups at both Eye Banks. This two-centre validation study showed that the use of RESEP increased the sensitivity of sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements. The test could not be validated without the use of RESEP.

Comparison of the Different Preservative Methods for Refractive Lenticules following SMILE

ABSTRACTPurposes: To (i) evaluate various methods for preserving refractive lenticules (RLs) from myopic eyes following small-incision lenticule extraction (SMILE), in order to (ii) establish a sound, standard storage RL preservative for clinical uses.Methods: In this prospective study, we compared freshly excised post-SMILE RLs (control group) with post-SMILE RLs (experimental group). Experimental group RLs were preserved in one of several preservatives: glycerol, allochroic silicagel desiccant, or Optisol. Following preservation in one of these three media, samples were evaluated by light microscopy (LM), and transmission (TEM) and scanning (SEM) electron microscopy on days-1, -3, -7, and -14.Results: Changes in cellular morphology were observed at all time points. Compared with fresh control-group RLs, there were significant histological changes in RLs preserved in glycerol and allochroic silicagel, but not Optisol. Comparison of the three methods revealed Optisol to be the best, followed by allochroic silica gel desiccant, followed by glycerol. RLs preserved in Optisol maintained the highest degree of viability and integrity. And the RLs viability and collagen density decreased with prolongation of storage time all.Conclusions: Optisol is a midterm corneal storage medium, which can maintain post-SMILE corneal RLs for 14 days, is a feasible and effective method for tissue storage.

Efficacy of Amphotericin B Against Fusarium and Aspergillus in Corneal Storage Medium.

The incidence of postkeratoplasty fungal infection is increasing in the United States, and our most commonly used corneal storage medium, Optisol-GS, contains antibiotics but no antifungal agents. We previously demonstrated the efficacy of amphotericin B additives in eliminating Candida albicans contaminants in Optisol-GS. The purpose of this study was to determine whether amphotericin B would also be efficacious against Fusarium solani and Aspergillus fumigatus. Vials of Optisol-GS were supplemented with 0.255 μg/mL of amphotericin B. Half of the vials were inoculated with F. solani and half with A. fumigatus. Positive control vials were inoculated with the fungi but no amphotericin B. The vials were refrigerated, sampled, and plated at different time points. The plates were then incubated at 36°C for 48 hr after which fungal colony counts were performed. There was an average reduction in the growth of F. solani in the amphotericin B-supplemented vials of 44% on day 2, 79% on day 7, and 80% on day 14 when compared with the positive control vials. There was an average reduction in the growth of A. fumigatus in the amphotericin B-supplemented vials of 40% on day 2 and 14% on day 7 when compared with the positive control vials. Both amphotericin B-supplemented and control vials grew less than 2 colonies of A. fumigatus on day 14. This study suggests that amphotericin B additives in Optisol-GS reduce the growth of F. solani and A. fumigatus.

Endothelial Cell Viability of Donor Corneas Preserved in Eusol-C Corneal Storage Medium.

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

Descemet Membrane Endothelial Keratoplasty Donor Preparation

The aim of this study was to describe the challenges in Descemet membrane endothelial keratoplasty (DMEK) donor preparations and provide new strategies to achieve success. A series of 263 consecutive DMEK preparation attempts by a novice surgeon during a corneal fellowship are described. In all cases, the Descemet membrane (DM) and the endothelium were peeled off from the donor cornea while it was submerged in corneal storage medium. The success rate of preparing DMEK tissue was 99%. Three donor preparations of 263 (1.1%) could not be completed successfully because spots of strong adherence between the DM and the stroma caused multiple horseshoe-shaped tears (HST) to form in the DM. Lamellar splitting of the DM ("partial thickness HST") preceded the formation of most HSTs. At least 1 HST occurred in 13% of donor preparations. In donor pairs (right and left corneas of 1 individual donor), if 1 cornea had any HSTs, there was a 60% chance that the contralateral cornea would have at least 1 HST. If 1 cornea had multiple HSTs, there was an 80% chance that the contralateral cornea would have at least 1 HST. Noting this trend, 3 donor corneas were returned to the eye bank unopened for other uses after their mates had multiple HSTs. With appropriate techniques, DMEK donor preparation can be highly successful, even for a novice surgeon. When a donor develops multiple HSTs, we recommend not using the mate for DMEK because of a higher risk of encountering a preparation difficulty.

A quantitative method to evaluate the donor corneal tissue quality used in a comparative study between two hypothermic preservation media

To standardize a new evaluation technique for calculating the overall quality (OQ) of the donor cornea and validate it using a comparative study of corneas preserved in Optisol-GS and Cornea Cold®. Thirty pairs of donor corneas were selected for a 4 week in vitro comparative study using masked observers. Physiological parameters like thickness, transparency, viable endothelial cell density (VECD) and morphology were transformed to numerical range (0-4) to obtain the OQ. Microbiological examination was performed using Bactec instrument. Students t test showed statistically better results (p < 0.05) from week 3 for thickness, week 2 for transparency and week 1 for morphology and VECD; statistical significance (p < 0.05) was found for OQ from week 2 for the corneas preserved in Cornea Cold® compared to Optisol-GS. Epithelial quality was similar regardless of the medium. Microbiological examination showed absence of aerobic and anaerobic microorganisms in both media. OQ method is efficient, consistent and easy, now validated for comparative studies. Further refinement is necessary for its use at eye-banks, bio-banks and research or transplantation purposes. Cornea Cold® is a promising hypothermic corneal storage medium with preservation time ≤21 days. This permits higher flexibility, evaluation accuracy, longer duration for surgical preparation and ease of transportation.

Quality assessment of corneal storage media and their components

To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.