Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

A novel human donor cornea preservation cocktail incorporating a thermo-reversible gelation polymer (TGP), enhancing the corneal endothelial cell density maintenance and explant culture of corneal limbal cells.

PURPOSE: McCarey-Kaufman's (MK) medium and Optisol-GS medium are the most commonly employed media for human donor corneal preservation. In this study, we evaluated the preservation efficacy of discarded human donor corneas using a Thermo-reversible gelation polymer (TGP) added to these two media. METHODS: Thirteen human corneal buttons collected from deceased donors, which were otherwise discarded due to low endothelial cell density (ECD) were used. They were stored in four groups: MK medium, MK medium with TGP, Optisol-GS and Optisol-GS with TGP at 4 °C for 96 h. Slit lamp examination and specular microscopy were performed. Corneal limbal tissues from these corneas were then cultured using explant methodology one with and the other without TGP scaffold, for 21 days. RESULTS: MK + TGP and Optisol-GS + TGP preserved corneas better than without TGP, which was observed by maintenance of ECD which was significantly higher in Optisol-GS + TGP than MK + TGP (p-value = 0.000478) and corneal thickness remaining the same for 96 h. Viable corneal epithelial cells could be grown from the corneas stored only in MK + TGP and Optisol-GS + TGP. During culture, the TGP scaffold helped maintain the native epithelial phenotype and progenitor/stem cell growth was confirmed by RT-PCR characterization. CONCLUSION: TGP reconstituted with MK and Optisol-GS media yields better preservation of human corneal buttons in terms of relatively higher ECD maintenance and better in vitro culture outcome of corneal limbal tissue. This method has the potential to become a standard donor corneal transportation-preservation methodology and it can also be extended to other tissue or organ transportation upon further validation.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

A new approach to extend the storage of donor corneas after 28 days of corneal culture in an Italian eye bank.

This study aimed to evaluate the possibility to extend the storage of unused organ-cultured donor corneas. After 28 days of corneal culture in TISSUE-C (AL.CHI.MI.A. S.R.L., Italy) and 5-day storage in transport/deswelling medium CARRY-C (AL.CHI.MI.A. S.R.L., Italy), 25 corneas that were deemed suitable for transplantation were transferred in fresh TISSUE-C at 31 °C for additional 7 days and then in fresh CARRY-C at room temperature for 24 h. Tissues were assessed for endothelial cell density (ECD), endothelial mortality and morphology after the standard and the extended corneal storage. In addition, the effect of donor age < 85 years and ≥ 85 years on corneal characteristics was assessed. After the extended storage, 6 out of 25 tested corneas (24%) showed ECD values below the acceptance limit (< 2000 cells/mm2). 19 corneas (76%) were still suitable for transplantation and showed a 5.9% loss in ECD, which was not statistically significant (P = 0.0949) compared to standard storage period. The two donor age groups did not show statistically significant differences in any tested parameter, although a trend for lower ECD and higher mortality in Descemet's folds after standard storage was observed in the ≥ 85 age donor group. Thus, the attempt of the current study to provide new sight-restorative options for unused tissues and increasing the availability of corneas in case of shortage gave encouraging results. Although a higher vulnerability of corneas from very old donors could not be statistically demonstrated in the present study, higher sample size could be required for prolonging the shelf life of these tissues.

In vitro susceptibility of microorganisms isolated from cold stored corneas: increased gentamicin-resistance in cornea banking.

Donor cornea contamination is one of the major risks for corneal transplants. The use of antibiotics in storage media remains as one of the most important security measurements to minimize the contamination risk in corneal preservation. Since antibiotic resistance among microorganisms have been rising gradually, it is important to gain knowledge about the antimicrobial susceptibility pattern for choosing the most suitable antimicrobial agents. Thus, we evaluated the in vitro susceptibility of microorganisms isolated in donor corneas processed at the Center for Blood Transfusion, Tissues and Cells (Córdoba, Spain) during 4 years in order to evaluate the efficiency, and to promote changes for further antibiotics use. Our results show the high rate of resistance to gentamicin, an antibiotic used in corneal preservation media such as Optisol GS and Eusol-C. Conversely, all the analyzed microorganisms were sensitive to vancomycin. This suggests the possibility of replacing gentamicin with another more effective antibacterial agent such as vancomycin.

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 10-100 cfu 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 for 7 days, 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.

Factors that influence the suitability of human organ-cultured corneas.

PURPOSE: The purpose was to assess the influence of donor and storage factors on the suitability of organ-cultured corneas for transplantation. METHODS: Data from 1340 donor corneas stored between 2009 and 2015 were analyzed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the suitability of grafts for transplantation. RESULTS: Forty-one percent (553/1340) of corneas were discarded. The leading causes were medical contraindication (20.2 %) and poor endothelial quality (19.3 %). Donor age influenced suitability for transplantation significantly. Corneas from donors aged 80 years and older were more likely to be discarded because of endothelial insufficiency (P < 0.0001). The cause of donor death including infection and multiple organ dsyfunction syndrom (MODS) increased the risk of bacterial or fungal contamination during organ culture (P = 0.007 and P = 0.014, respectively). Prolonged time between death and enucleation was associated with an increased risk of unsuitability for transplantation (P < 0.0001). The amount of time between death and corneoscleral disc excision and duration of storage influenced the suitability for transplantation (P = 0.0007 and P < 0.0001, respectively). CONCLUSION: Donor age, cause of death, storage time, death to enucleation and death to disc excision times influenced transplantation suitability. The percentage of discarded corneas may be reduced by shortening storage time, death to enucleation, and death to corneoscleral disc excision times. Setting a maximum donor age could reduce the percentage of discarded corneas. However, as long as there is a lack of donor corneas, we do not recommend any donor age limit.

Donor related corneal graft infection: a review of literature and preventive strategies.

PURPOSE: Donor-related infections are a serious threat to patient safety after corneal transplantation. We provide a concise review of literature from the last decade on donor-related graft infections, sources of contamination and means to reduce the contamination of donor tissue and preservation media. METHODS: We reviewed 50 papers from year 2005 to 2021 related to donor-related graft infections. We included 14 studies related to the risk factors associated with post-keratoplasty infection and preventive methods. RESULTS: Incidence of post-keratoplasty infections has been reported to be approximately 0.2%-0.77% for endophthalmitis and 6.5%-10.5% for microbial keratitis. We analyzed six important studies regarding the risk factors related to donor contamination. It was observed that younger donor age, increased death to retrieval time, warming cycles and increased eye bank processing time and positive corneo-scleral rim cultures were important risk factors for donor-related infections post keratoplasty. Eye banks have adapted newer protocols over the time period for prevention of donor-related contamination. Recommended preventive strategies were published in about eight important studies over the past decade. In addition to meticulous donor screening, rapid warming cycles, double contact with povidone iodine during retrieval and addition of antifungals like amphotericin B, Voriconazole and cycloheximide have been suggested over the last decade although their use is still in debate with regard to the efficacy, toxicity and cost-effectiveness. CONCLUSION: The last decade has witnessed a relative rise of fungal infections and multidrug resistant bacterial infections post-keratoplasty. Eye bank prepared corneas for lamellar surgeries are at increased risk for donor contamination due to increased exposure to the higher temperatures during their processing. Addition of antifungals and broad spectrum antibiotics to the hypothermic preservation media needs to be considered in the new era of increasing trends of lamellar keratoplasty.

Allogenic Lenticular Implantation for Correction of Refractive Error and Ectasia: Narrative Review.

INTRODUCTION: Intrastromal lenticule implantation is a promising treatment option for corneal pathologies, from refractive error to ectasia. In this narrative review, we intend to feature up-to-date literature supporting the use of lenticular tissue, a compelling method that can be customized for a variety of applications, providing an additional source of donor tissue for treating corneal diseases. METHODS: We searched databases PubMed, Mendeley, and Scopus last accessed 10 May 2023, for literature on stromal lenticules and narrowed based on relevance. Review articles, animal studies, ex vivo studies, and book chapters were excluded, while assessable and relevant articles published in English were included. RESULTS: Storage methods from using fresh lenticules to dehydration have proven successful, with cryopreservation maintaining structure and cellular viability for up to 10 years. Successful use of lenticules for treatment of numerous pathologies including corneal ectasias, hyperopia, and presbyopia with additional insight into the treatment of corneal ulcers and perforations are highlighted in this narrative review. CONCLUSION: Lenticular implantation is an innovative and advantageous treatment for various ocular pathologies, offering increased bioavailability, flexibility, and customization for patients. They can treat previously untreatable diseases and serve as a replacement for synthetic implants, with promising outcomes worldwide. Lenticular implantation has the potential to become a leading approach in ophthalmologic surgery. Further studies should aim to provide evidentiary support for a standardization of lenticule banking.

The importance of donor characteristics, post-mortem time and preservation time for use and efficacy of donated corneas for posterior lamellar keratoplasty.

PURPOSE: The aim of this study is to examine whether donor age, death-to-retrieval time (DRT) and death-to-preservation time (DPT) as well as total preservation time affect donor cornea suitability for endothelial keratoplasty (EK) or penetrating keratoplasty (PK). METHODS: A registry-based study was performed identifying 3248 corneas donated between 2011 and 2017. Data regarding donated corneas were extracted from The Danish Cornea Bank and donor medical records and evaluated for missing information. The primary outcome was whether ECD at preservation (ECD-P) or at release (ECD-R) was >2000 cells/mm2 . RESULTS: Logistic regression for ECD-P showed a significant negative effect of increasing age (OR: 1.07, 95%CI: 1.05;1.08, p < 0.001) on donor suitability. Higher ECD-P had a significant positive effect on graft eligibility (OR: 1.007 95%CI: 1.003;1.010, p < 0.001). No significant effect of donor sex (p = 0.547), DRT (p = 0.289) or DPT (p = 0.102) on donor suitability for EK or PK (Chi-squared test). CONCLUSION: High donor age and low ECD-P negatively affect the suitability of donor corneas for EK/PK whereas DRT and DPT did not affect graft suitability.

Evaluation of various preservation media for storage of donor corneas.

PURPOSE: To compare the physical and microbiological characteristics of McCarey-Kaufman (MK), Cornisol, and Optisol-GS media and evaluate the outcomes of keratoplasty performed using corneas stored in these three media. METHODS: The study involved 60 donor corneas which were distributed in 3 groups: MK, Cornisol, and Optisol-GS. Corneas in these groups were further analyzed based on the type of keratoplasty performed (full thickness versus endothelial keratoplasty). At baseline, the endothelial cell density and death to preservation time of donor corneas were recorded. Following keratoplasty, patients were evaluated on day 1, at 1 month, 3 months, and 6 months follow-up. Outcomes were assessed in terms of corrected distance visual acuity (CDVA), endothelial cell density, percentage endothelial cell loss, and corneal thickness. The storage media were also assessed for their physical quality and their microbiological characteristics. RESULTS: Physical characteristics of all three media were found to be within normal limits. Mean CDVA was comparable among the 3 groups at 6-month follow-up. The absolute endothelial cell count values were significantly lower for corneas stored in MK medium (1873.7 ± 261.1 cells/mm2) compared to the Cornisol (2085.0 ± 230.3 cells/mm2) and Optisol-GS media [(2180.3 ± 217.2 cells/mm2) (P = <0.001)]. Corneas stored in Optisol-GS medium were significantly thinner at 1-month follow-up with no significant difference at 6 months (P = 0.66). CONCLUSION: Optisol-GS and Cornisol media were found to preserve endothelial cell density better and stabilize corneal thickness earlier as compared to the MK medium. However, the functional outcomes were comparable among the three groups.

In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting.

Factors influencing the contamination rate of human organ-cultured corneas.

PURPOSE: To assess the influence of donor, environment and storage factors on the contamination rate of organ-cultured corneas, to consider the microbiological species causing corneal contamination and to investigate the corresponding sensitivities. METHODS: Data from 1340 consecutive donor corneas were analysed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the contamination rate of organ-cultured corneas for transplantation. RESULTS: The mean annual contamination rate was 1.8 ± 0.4% (range: 1.3-2.1%); 50% contaminations were of fungal origin with exclusively Candida species, and 50% contaminations were of bacterial origin with Staphylococcus species being predominant. The cause of donor death including infection and multiple organ dysfunction syndrome increased the risk of bacterial or fungal contamination during organ culture (p = 0.007 and p = 0.014, respectively). Differentiating between septic and aseptic donors showed an increased risk of contamination for septic donors (p = 0.0020). Mean monthly temperature including warmer months increased the risk of contamination significantly (p = 0.0031). Sex, donor age, death to enucleation, death to corneoscleral disc excision and storage time did not increase the risk of contamination significantly. CONCLUSION: The genesis of microbial contamination in organ-cultured donor corneas seems to be multifactorial. The main source of fungal or bacterial contamination could be resident species from the skin flora. The rate of microbial contamination in organ-cultured donor corneas seems to be dependent on the cause of donor death and mean monthly temperature.

Full-field optical coherence tomography of human donor and pathological corneas.

PURPOSE: To evaluate the performance of a full-field optical coherence tomography (FF-OCT) system in the study of human donor and pathological corneas and assess its suitability for use in eye banks. METHODS: Our study was carried out using an FF-OCT system developed for non-invasive imaging of tissue structures in depth with ultrahigh resolution (1 µm in all directions). Images were acquired from eight stored human donor corneas (either edematous or after deswelling) and five surgical specimens of corneas with various diseases (bullous keratopathy, lattice corneal dystrophy, stromal scar after keratitis, keratoconus and Fuchs dystrophy). They were compared with standard histology and pre-operative spectral domain OCT. RESULTS: The FF-OCT device enabled a precise visualization of the cells and the different structures (epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane and endothelium) in normal corneas. Specific lesions in various corneal diseases could also be easily identified, such as corneal edema, epithelium and Bowman's layer irregularities, breaks, or scars (keratoconus), stromal opacities, deposits, fibrosis (stromal corneal scar, bullous keratopathy, lattice corneal dystrophy) and Descemet's membrane thickening and guttae (Fuchs dystrophy). FF-OCT image features were comparable to the details provided by conventional histology. Higher resolution could be demonstrated with FF-OCT when compared with spectral domain OCT. CONCLUSION: FF-OCT is a powerful non-invasive imaging tool that allows detailed study of corneal structures. Images correlate well with conventional histology. Further studies should evaluate the benefit of this technique as a complement to current assessment methods of human donor corneas.

The suitability of corneas stored by organ culture for penetrating keratoplasty and influence of donor and recipient factors on 5-year graft survival.

PURPOSE: To determine the impact of donor factors on the suitability of corneas stored by organ culture for penetrating keratoplasty (PK) and the influence of donor and recipient factors on 5-year survival of first PK. METHODS: Logistic regression analyses were carried out to determine the influence of donor factors on, respectively, the risk of microbial contamination during organ culture, the suitability of corneas for PK (endothelial cell density ≥ 2200 cells/mm(2)), and the quality of corneas (endothelial cell density ≥ 2500 cells/mm(2)). Only one cornea, randomly selected, from each donor was included in these analyses. A Cox regression analysis was used to determine the influence of donor and recipient factors on 5-year PK survival. RESULTS: Risk of contamination (n = 8317): Causes of donor death including infection, respiratory disease, and cancer all increased the risk of contamination during organ culture (P < 0.0001). Suitability for PK and endothelial quality (n = 7107): Donor age (P < 0.0001) and storage time in organ culture (P < 0.0001) were the principal factors affecting suitability and quality. Death to enucleation and enucleation to processing times had little influence. Corneas from organ donors were more likely to be suitable for PK (P = 0.0003). Five-year graft survival (n = 3014): Graft survival was dominated by the indication for PK (P < 0.0001). Allograft rejection was also a major risk factor for failure (P < 0.0001). The only donor factor affecting survival was sex (P = 0.008). CONCLUSIONS: Donor age and storage time but not postmortem times influenced the suitability of corneas for PK. The indication for PK and other recipient factors were the main predictors of graft failure.