Organ Culture Storage Research Articles

Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C.

In this experimental study, we compared the structural integrity and cell quality of corneal endothelium stored in organ culture medium (OCS) and Eusol-C. The experiment included rabbit and human cornea experiments in vitro. Thirty rabbit corneas and thirty-two human corneas were collected and divided into two groups. All right corneas were allocated in experiment group and left corneas were placed in control group. The corneas in experimental group were stored in OCS at 34°C, and the corneas in control group were stored in Eusol-C at 4°C for 7, 14, 21, 28, and 35days, respectively. Endothelial cell morphology, cell count, and trypan blue staining for viability were assessed before storage (Day 0) and at days 7, 14, 21, 28 and 35. The structural integrity of human corneal endothelial cell was analyzed using immunohistochemistry. The samples of storage solution for microbial culture were collected on the third day and at the end of storage. The results show that no bacterial and fungal infections were found in both groups. After 14days of storage, the morphology of endothelial cell was better in the experimental group than in the control group. The endothelial cell stored in OCS were better than those stored in Eusol-C at the end of storage times, except human cornea 14days storage group. The ZO-1 protein staining showed the typical polygonal morphology of endothelial cell stored in the OCS. Corneal endothelial cells stored in the OCS had better quality up to 28days. It can be applied to Chinese eye banks as a method of corneal preservation.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell–matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Overview of Short-Term and Intermediate-Term Corneal Storage Solution: Comparison of Clinical Outcomes and Need For Future Research.

Corneal donor tissue preservation techniques have incrementally improved since the introduction of McCarey-Kaufman storage solution from short-term storage to intermediate duration of storage with the advent of organ culture and Optisol GS storage solutions. Improved understanding of the corneal endothelial cell physiology has helped in designing newer storage solutions, such as the Life 4C and Cornea Cold. The incorporation of antibiotics, ATP precursors, minerals, and vitamins has improved the viability of tissues. In addition, these modifications to the newer storage solutions have increased the endothelial longevity and metabolic activity. Despite these advances, the duration of tissue storage has largely been restricted to 2 weeks in Optisol GS and 4 weeks in organ culture. The role and cost-effectiveness of antifungal supplementation and the need for improved epithelial preservation are additional areas that need to be explored. This review intends to summarize the efficacy and viability of donor corneas in different tissue storage solution and compare clinical outcomes while providing an insight into the challenges in developing newer methods of corneal preservation.

Preservation of corneal stromal lenticule: review.

Corneal stromal lenticule is a part of corneal stroma, which can be created by manual dissection, by femtosecond laser from the donor cornea, but chiefly it is a waste product of a refractive procedure ReLex SMILE (Small Incision LenticuleExtraction). Corneal lenticule has a huge potential in corneal surgery. In recent years, many studies have been published to show the possibility to use this tissue to treat corneal defects, as well as in refractive surgery. Thanks to the quantity of lenticules which arise every day during SMILE operations, this tissue is much more accessible than any other kind of corneal tissue. According to the experience with lenticule implantation in animal models, or even human patients, lenticule implantation is considered safe, reversible method, which is not associated with immune rejection or other severe complications. However, the crucial step before the process of lenticule implantation, is proper preservation of this tissue. Donor corneal tissue containing endothelium is usually preserved in hypothermia and then usable maximally for two weeks. Newer methods such as organ culture storage and use of a sterile cornea prolong the time of usability of the tissue. The possibilities for corneal lenticule storage are theoretically wider thanks to the fact, that we do not need to preserve fragile cellular structures. Besides the storage in hypothermia, other preserving methods such as cryopreservation and storage after decellularization have been tested. This review aimed to examine the current literature that describes possible methods of corneal lenticule preservation. A comprehensive search was created based on articles published in English on PubMed.gov, Cochranelibrary.com and Scopus.com using following keywords: corneal lenticule preservation, corneal lenticule storage, cold storage corneal lenticule, corneal lenticule cryopreservation till 2020.

Endothelial Cell Viability after DMEK Graft Preparation

ABSTRACT Aim: To evaluate the effect of graft preparation and organ-culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts. Materials and methods: DMEK grafts (n = 27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the Covid-19-related cancellation of elective surgeries. Cell viability (by Calcein-AM staining) and ECD of five grafts originally scheduled for transplantation were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post-preparation or after 3–7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein-AM staining (Calcein-ECD). Results: Light microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein-ECD for the five grafts initially allocated for transplantation was 18% (range 92–73%) lower than median LM ECD. For the paired DMEK grafts, Calcein-ECD determined by Calcein-AM staining on the day of graft preparation and after 3–7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3–7 days of graft storage was 88% and 92%, respectively. Conclusion: Cell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation, with insignificant additional ECD changes during 3–7 days of graft storage. Implementing an additional post-preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications.

Changes of Contamination Rate and Microorganism Evaluation in Organ-Cultured Human Corneas: A 14-Year Review From a French Regional Eye Bank.

This study aimed to assess how the contamination rate of organ-cultured corneas has evolved and to analyze the evolution of microorganisms involved. Data from the Besançon eye bank were reviewed over 14 years (2005-2018). The changes in the contamination rate and the contaminant species found during the organ culture storage were analyzed. Microbiological tests were performed twice on the storage media-at day 5 and before the deswelling phase. Among the 17,979 donor corneas collected, 1240 corneas were microbiological-test positive. The average annual contamination rate was 6.8% (range: 5.2%-8.9%). Seventy-five percent of contaminations were bacterial. The most frequently found bacterium was Staphylococcus spp. (31.3%), followed by non-Enterobacteriaceae Gram-negative Bacilli (GNB) (27.3%), with most Sphingomonas spp. and Pseudomonas spp. Fungal contamination (21.9%) was dominated by Candida (82.7%). Seventy-seven types of microorganisms were identified. The Staphylococcus rate tended to decrease, whereas non-Enterobacteriaceae GNB rate has increased in the past few years to reach 46% of bacteria. Most of the contaminations were detected in the early phase of organ culture at day 5 (89.2%). The second microbiological test found 44.8% of fungal contaminations (predominantly Candida spp.). The annual contamination rate was stable and remains low, but the types of contaminating microorganisms varied from year to year. Staphylococcus spp. and non-Enterobacteriaceae GNB accounted for a significant proportion of the contaminations. We found a significant proportion of contamination, especially fungal, at the late phase of storage. Reassessing the antibiotics and antifungals in the storage medium may be useful to limit corneal disposal.

The influence of preparation and storage time on endothelial cells in Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) grafts in vitro.

Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) has been introduced as a modification of the standard DMEK technique to increase the pool of endothelial grafts. In this study, we evaluated in vitro changes in endothelial cell distribution, viability and morphology of Quarter-DMEK grafts when stored in organ-culture medium. Quarter-DMEK grafts were prepared from 5 corneas and stored in organ-culture medium for 4, 7 and 11days. Endothelial cell re-distribution was investigated by light microscopy, cell viability by a Calcein-AM assay, and expression of endothelial and non-endothelial markers by immunohistochemistry. Three standard DMEK-grafts were used as controls. After preparation, all Quarter-DMEK grafts showed a band with no viable endothelial cells along the radial cut graft edges [average width 190 (± 20)µm]. Endothelial cell density in the central graft area decreased by 12%, 23% and 26% after 4, 7, and 11days of storage, respectively. At the same time, empty bands along the cut edges were re-populated and some cells migrated to the stromal side of the Descemet membrane (DM). These cells showed an altered phenotype, as indicated by expression of migration marker CD73 and fibroblast marker αSMA. Majority of migration occurred within the first 4days of storage. Our data suggest that endothelial cells on Quarter-DMEK grafts re-distribute during organ-culture storage to re-populate preparation-induced empty bands and after re-distribution, cells may show further migration to the stromal DM side during storage.

Factors Affecting Corneal Organ Culture Contamination: A 6-year Study at the New South Wales Tissue Bank.

To report the rate of microbial contamination and analyze possible risk factors for contamination of banked corneas stored using the organ culture method. Data from the New South Wales Tissue Banks incorporating the Lions NSW Eye Bank, between September 1, 2011, and November 30, 2017, were reviewed retrospectively. All corneas collected during this period and stored in organ culture storage media were tested for microbial contamination. The influence of potential factors on the rate of contamination was analyzed using the χ test and logistic regression using generalized estimating equations. A total of 4410 corneas were included in this study, of which 110 were medium culture positive, representing a microbial contamination rate of 2.5%. The main contaminants were Candida species followed by Staphylococcus species. Corneal tissue collected in summer and autumn had a significantly higher contamination rate (P = 0.006). All other factors studied were not shown to have a statistically significant association with contamination after accounting for within-pair correlation and confounders. A relatively low contamination rate of 2.5% observed in our study reflects the stringent laboratory protocols, strict donor selection criteria, and high level of experience among staff at the Lions NSW Eye Bank. Our study demonstrated that the season of collection had a strong association with the rate of organ culture contamination. Because Candida species contributed the largest percentage of contamination, specific measures to reduce and eliminate fungal proliferation should be considered by eye banks particularly in warm seasons.

Outcome of Human Donor Corneas Stored for More Than 4 Weeks.

Organ culture is the most common method for preservation of human donor corneas within Europe, and the maximum accepted storage period is typically set to 4 weeks. However, little is known about the fate of prolonged (4+ wks) organ culture storage of donor corneas. The aim of this study was to evaluate the clinical outcomes of corneal grafts preserved from 4 to 7 weeks compared with corneal grafts preserved up to 4 weeks in organ culture medium. A registry study was performed, identifying 1252 corneal transplantations [Descemet stripping automated endothelial keratoplasty (DSAEK) and penetrating keratoplasty (PKP)] between February 2008 and November 2015 at Aarhus University Hospital, Denmark. Data were extracted from The Danish Cornea Bank Registry and The Swedish Cornea Registry, which also includes Danish patients. The outcomes evaluated, 2 years after transplantation, were graft function, rebubbling rates in DSAEK surgeries, rejection rates, and endothelial cell density (ECD). A total of 808 donor corneas were stored for ≤29 days, with a mean preservation time being 20 ± 6 days (mean ± SD, range: 6-29 days). A total of 444 donor corneas were stored for >29 days, with a mean preservation time being 38 ± 7 days (mean ± SD, range: 30-72 days). In both DSAEK (n = 835) and PKP (n = 417), no statistically significant difference was observed in the frequency of graft failure (DSAEK, P = 0.9 and PKP, P = 0.74), rebubbling rates (DSAEK P = 0.07), rejection rates (DSAEK, P = 0.08 and PKP, P = 0.95), or ECD (DSAEK, P = 0.56 and PKP, P = 0.44) between donor corneas cultured for ≤29 days and donor corneas cultured for >29 days. Corneas stored in organ culture for more than 4 weeks are equally suitable for DSAEK and PKP transplantation as corneas stored in organ culture for up to 4 weeks.

Organ culture storage of pre-prepared corneal donor material for Descemet's membrane endothelial keratoplasty

PurposeTo evaluate the effect of media composition and storage method on pre-prepared Descemet's membrane endothelial keratoplasty (DMEK) grafts.Methods50 corneas were used. Endothelial wound healing and proliferation in different media were...

Implementation of Organ Culture storage of donor corneas: a 3year study of its impact on the corneal transplant wait list at the Lions New South Wales Eye Bank.

Organ Culture corneal storage offers an extended storage time and increased donor pool and tissue assessment opportunities. In September 2011, the Lions New South Wales Eye Bank (LNSWEB) moved from hypothermic storage to Organ Culture corneal storage. This study evaluates the impact of implementation of Organ Culture on donor eye retrieval and the corneal transplant waiting list over a 3year period in NSW, Australia. Retrospective review of the LNSWEB data from September 2011 to August 2014. Tissue collection, waiting list and tissue utilization data were recorded. The data from September 2008 to August 2011 for Optisol-GS storage was used for comparison. The annual donor and corneacollection rate increased 35% and 44% respectively with Organ Culture compared to Optisol-GS storage. The utilization rate of corneal tissue increased from 73.4% with hypothermic storage to 77.2% with Organ Culture storage. The transplant wait list decreased by 77.3% from September 2011 to August 2014 and correlated with the increased rate of corneal transplantation (r=-0.9381, p<0.0001). No other factors impacting the wait list changed over this period. Corneas not used from either storage method were due to unacceptable endothelial cell density/viability. The contamination rate of corneas stored in Organ Culture medium was low at 1.74%. The Organ Culture storage method increases the corneal donor pool available to Eye banks. The practical benefits of the extended storage time and increased donor assessment opportunities have directly led to an increase in corneal utilization rate and a significant decrease in recipient wait list time.

Dehydration of corneal anterior donor tissue with polyethylene glycol (PEG)-enriched media.

Anterior donor grafts (including scleral rim, without Descemet membrane) increase in thickness and become hazy upon storage in organ culture (OC) medium. Transfer of these grafts to standard dehydration media just before transplantation does not reduce their thickness to normal. Therefore, we assessed the efficacy of different media enriched with polyethylene glycol (PEG) as dehydrating agents for organ-cultured anterior donor grafts. Grafts were harvested and stored in the commercial OC medium 'Max' (without dextran) for 1 week, and subsequently dehydrated in the standard commercial dehydration medium 'Jet' (with dextran) supplemented with 4-20% PEG3350, or 'Max' supplemented with 20% PEG6000 and PEG20.000, or 5-20% PEG35.000. Central corneal thickness (CCT), as assessed by anterior segment-optical coherence tomography, and transparency were evaluated before, and at 1, 4 and 7 days of dehydration. Transfer of grafts after 1 week of OC (average 1,200 µm) to 'Jet' supplemented with PEG3350 revealed a concentration-dependent effect of dehydration; CCT was restored to normal (500-600 µm) when 10% PEG3350 was added. However, transparency was only temporarily restored; after 1 day, the grafts turned hazy. In contrast, grafts transferred to 'Max' supplemented with 20% PEG35.000 were transparent throughout the evaluation period, but were dehydrated to beyond normal levels (average 300 µm). 'Max' supplemented with 5% PEG35.000 dehydrated grafts to normal values and restored transparency throughout. Thus, dehydration of anterior donor grafts prior to surgery in dextran-free OC medium supplemented with 5% PEG35.000 reduces graft thickness to normal and may facilitate anterior keratoplasty procedures.

DMEK lenticule preparation from donor corneas using a novel ‘SubHyS’ technique followed by anterior corneal dissection

PurposeTo describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas.Methods30 human...

Novel Technique for the Preparation of Corneal Grafts for Descemet Membrane Endothelial Keratoplasty

To report a simple novel technique to facilitate preparation of Descemet membrane grafts for Descemet membrane endothelial keratoplasty (DMEK). Laboratory investigation and retrospective, single-center, consecutive case series. Preparation of the endothelial graft is performed on an artificial anterior chamber, endothelial side up. After an incomplete circular superficial trephination, we describe a simple technique using a 27 gauge cannula to detach the Descemet membrane (DM). Endothelial cell density (ECD) was measured before dissection on 12 human corneas for research and 3 days after storage in organ culture. Histologic and electron microscopy analysis were performed. A DMEK was performed in 50 patients with Fuchs dystrophy. Visual acuity and ECD were evaluated 2 and 6 months after surgery. ECD was 2765 ± 256 cells/mm(2) on corneas for research before dissection and 2651 ± 305 cells/mm(2) after 3 days in organ culture (P < .01). Histologic and electronic sections confirm that the cleavage was between DM and posterior stroma. Clinically, preparation of 2 corneas from a single donor was unsuccessful; 48 corneas were clear at 2 months and 47 at 6 months. At 2 months 77% of the patients had recovered a visual acuity of at least 20/30. At 6 months, 91.5% of the patients had a visual acuity of at least 20/30. ECD was 2656 ± 28 cells/mm(2) (range: 2450-3100 cells/mm(2)) preoperatively, 1797 ± 41 cells/mm(2) (range: 1100-2700 cells/mm(2)) at 2 months, and 1658 ± 43 cells/mm(2) (range: 900-2600 cells/mm(2)) at 6 months. We report here a reliable and efficient technique for the preparation of pure Descemet membrane grafts.

Simple novel surgical device to facilitate preparation of endothelial grafts for DMEK

AbstractPurpose Descemet’s membrane and endothelial keratoplasty (DMEK) is the only technique to achieve a posterior corneal transplant without any interface. It is however difficult to achieve, mainly during the preparation of the graft. We propose a simple novel surgical device to facilitate this preparation.Methods We used 20 corneas for research in this study. The device is in the form of a circular blade with a diameter of 8.5 mm. The blade is broken in two opposite points on 4 mm. The blade is applied to the cornea in order to cut Descemet's membrane and deep stroma. Preparation of the endothelial graft is performed on an artificial anterior chamber using a simple technique with a 27 gauge cannula to detach Descemet’s membrane. The dissection is performed under the microscope by a corneal bank technician in 8 cases, by a resident in 4 cases by a cataract surgeon in 8 cases. The three operators followed a specific course on the same day of dissection but had never tried to dissect a DMEK graft before. Endothelial cell density (ECD) was measured before dissection and 3 days after storage in organ culture. Histological analysis was performed.Results Descemet graft could be easily dissected in 19 cases and resident failed dissection in one case. ECD was 2534±275 cells/mm² before dissection and 2412+/‐360 cells/mm², after 3 days in organ culture (p&lt;0.01). Histological sections confirm that the cleavage was between DM and posterior stroma.Conclusion We report here a reliable and efficient technique for the preparation of pure descemetic grafts. Commercial interest

Hypothermic storage or organ culture?

AbstractCurrently there are two storage practices for the cornea, both use liquids based on cell culture medium: the hypothermic storage at 2‐6°C, and organ culture at 30‐37°C. Hypothermic storage seems to offer donor tissues of good quality comparable to that obtained by organ culture, provided that the storage time is kept short. Indeed, according to the literature, the risk of primary graft failure increases significantly after storage longer than 7 days. Furthermore, corneas stored longer than 7 days display epithelial alterations that may hinder the surgical procedure or delay the full recovery of the graft. The organ culture storage method consists of a storage period in culture medium at 30‐37C°, and a shorter de‐swelling and transportation phase at 30‐37C° and room temperature in the same medium supplemented with 4‐8% dextran. Organ culture solutions contain fetal calf serum as a source of growth factors to limit endothelial cell loss. The serum must be obtained from prion‐free countries. A storage period of 30 days can be achieved without significant loss of endothelial cells. The endothelium shows reparative phenomena during storage. Organ culture offers a longer storage time, a less restricted donor supply on beforehand, corneal endothelium with a better defined quality, and a pre‐operative microbiologic control. Cultured corneas always display an epithelium made up of 2‐3 layers of viable cells. The 30 days storage period allows an efficient use of valuable donor tissue: planning of operations is easier, allowing sufficient time for the allocation. The disadvantages of this method are the relative technical complexity and the need for qualified staff to perform tissue culture and selection of the corneas. The studies comparing the effect of the storage methods on outcome demonstrate similar graft survival and post‐operative decline in endothelial cell density. Corneas stored by organ culture are at least comparable with those stored hypothermically for shorter periods.

An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants

Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo-scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo-scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth.

Light microscopy of the corneal pathologies that we meet in our daily practice as an eye banker

AbstractPurpose Organ culture is the method of choice for the preservation of a human donor cornea for transplantation throughout Europe. This method is often closely connected with the assessment of the cornea using light microscopy. The corneal endothelium, its polymeghatism, pleomorphism, cornea guttata and other endothelial abnormalities as well as epithelial and stromal pathologies will be presented to show the usefulness of light microscopy in the evaluation of corneal pathologies.Methods Photographs from light microscopy (both phase contrast and bright field) taken before and after corneal storage in organ culture will be compared and discussed in relation to changes in endothelial morphology and the swelling of the intercellular spaces. The individual corneal layers (the corneal epithelium, the stroma and the endothelium) of pathological corneas will be compared to those of control corneas.Results Among corneal pathologies, cornea guttata, corneal dystrophies (Fuchs endothelial corneal dystrophy, posterior polymorphous corneal dystrophy) and crystalline keratopathy will be shown using pathological explants. Moreover, the corneal pathologies will be correlated with histological findings. Finally, bacterial and fungal contamination of the cornea during organ culture will be shown.Conclusion Light microscopy is an essential part of qualitative and quantitative corneal assessment, which allows corneas with a variety of pathologies, mostly endothelial in origin, to be excluded from grafting. This work was supported by the project PRVOUK‐P24LF13 of Charles University in Prague.

Rabbit, rat and pig corneas: main characteristics and storage in organ culture

AbstractPurpose Except for primates, animal models of corneal are far from human. Animal models of corneal storage are virtually non‐existent. Aim: to update the main characteristics (especially for endothelial cells (EC)) of the cornea of 3 easily available animals, as well as their ability to be stored in organ culture (OC)Methods 30 corneas of 6 month‐old Large White pig, 20 of 10 week‐old California rabbits and 10 of 8 week‐old Lewis rats were investigated. Macroscopic data: 1/horizontal and vertical diameters (digital calliper), 2/transparency (analysis of modulation and contrast transfer functions), 3/ corneal thickness (CT) (ultrasound pachymetry). Microscopic data: 1/ histology on Hematein‐Eosin‐Safran stained cross sections and ultrastructure, 2/ EC density (ECD) and morphometry, 3/ EC proliferative status (Ki67 and 5‐Ethynyl‐2’‐Deoxyuridine incorporation (Clik‐it EdU)), differentiation status (Na+/K+ ATPase, ZO‐1, JAM‐1), and existence of stem cells (Nestin, ABCG2, Telomerase). Fresh corneas were organ cultured in 2 commercially available media. CT was measured every 2h during 6h, then every 12h. Transparency and EC survival was determined after 2, 3 and 4 days of OCResults We constituted a complete database of ex vivo corneal characteristics of the 3 species. ECD, polymorphism and polymegathism were higher than in human. CT increased rapidly (up to 3 times) in both OC media, resulting in biconvex tissues with almost complete loss of transparency and significant endothelial folding. EC survival decreased especially in foldsConclusion Corneas of the 3 animals cannot be stored more than 2 to 3 days in OC media designed for human. Specific media should be developed to obtain reliable models of animal corneal OC

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.