Human Organ-cultured Corneas Research Articles

How Implementing a Quality Management System at the LIONS Eye Bank Saar-Lor-Lux, Trier/Western Palatinate from 2006 to 2016 Impacted the Rate and Reasons for Discarding Human Organ-Cultured Corneas

The aim of this study was to explore how the implementation of the quality management system (QM; according to DIN EN ISO 9001 : 2008) at the LIONS Eye Bank impacted the rate and reasons for discarding donated corneas. The QM system was introduced in November 2010. The rate of and reasons for discarding cornea tissues at the LIONS Eye Bank Saar-Lor-Lux,Trier/Western Palatinate from 2006 to 2016 were analysed retrospectively. 4,140 corneas from 2,084 donors were analysed. 1,640 corneas were processed before the QM system had been introduced and 2,500 corneas thereafter. Main reasons for discarding cornea tissues were endothelial quality, contamination of the medium and a positive conjunctival swab. The data was extracted from clinical files and data filing systems, entered into a Microsoft Access database and analysed statistically with SPSS. Following the implementation of a QM system, the discard rate decreased significantly from 50.1% to 39.7% (p < 0.0001). Prior to the implementation of the QM system, 493 (30.1%) cornea tissues had been discarded due to endothelial quality, while this figure improved significantly to 604 (24.2%) after the QM system had been implemented (p < 0.0001). Contamination was the reason for discarding tissue in 173 (10.5%) cases before implementation of the QM system, and in 124 (5.0%) cases thereafter (p < 0.0001). Positive conjunctival swab was the reason for discarding tissue in 10 (0.6%) cases before the QM system had been implemented, and in 53 (2.1%) cases thereafter (p < 0.0001). Implementation of a QM system significantly reduced the rate of discarded donor tissue in the LIONS Eye Bank. Better management of contamination as a reason for discarding cornea tissues can be attributed to improved standards, protocols and training that are part of this QM system.

Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus

Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

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

The purpose was to assess the influence of donor and storage factors on the suitability of organ-cultured corneas for transplantation. 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. 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). 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.

Femtosecond laser cutting of multiple ultrathin corneal stromal lamellae for endothelial graft bioengineering

AbstractPurpose The bioengineering of endothelial grafts (EG) is developing in several countries as an answer to the shortage of donor corneas worldwide. They are composed of a thin transparent biocompatible carrier evenly covered with tightly packed fully functional endothelial cells. Multiple options exist for the carriers. In our laboratory, we are focused on the optimization of human donor tissue because the well‐organised cornea donation in France is not the most limiting point. Aim: to present the preparation of ultrathin stromal lamellae from a single human corneaMethods Human organ‐cultured corneas unsuitable for graft were used either with our without deswelling in Dextran containing medium. They were mounted on a artificial anterior chamber and cut with a femtosecond laser (femto LDV Z6, Ziemer) with aplanation. Six horizontal parallel cuts were realized on a 9 mm diameter area without vertical section. Cornea was then trephine at 8.00mm on a punch trephine. Thickness map was obtained before and after the cut with an AS‐OCT equipped with a custom adapter for ex vivo corneas. Lamellae were separated under an operating video‐microscope and again observed with OCT. Quality of surface were analysed with scanning electron microscopy and multiphotonic confocal microscopy with SHG for collagen fiber imagingResults Seven regular lamellae of approximately 100μm were obtained on a single cornea and easily detached. Characterisation of surface quality is ongoingConclusion Femtosecond laser cutting of multiple lamellae on a single human cornea could allow obtaining a sufficient number of thin transparent and fully biocompatible carriers for EG bioengineering

Normalization of Wound Healing and Diabetic Markers in Organ Cultured Human Diabetic Corneas by Adenoviral Delivery ofc-MetGene

Purpose. Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor (HGF) receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV)-driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities. Methods. Forty-six human corneas obtained postmortem from 23 donors with long-term diabetes (5 with diabetic retinopathy) were organ cultured and transduced with rAV-expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at approximately 10(8) plaque-forming units per cornea for 48 hours. Each control fellow cornea received control rAV (rAV expressing the beta-galactosidase gene or vector alone). After an additional 4 to 5 days of incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. The corneas were analyzed afterward by immunohistochemistry and Western blot analysis. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blot analysis, and inhibitor analysis. Results. rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular, and phosphorylated) and normalization of the patterns of select diabetic markers compared with rAV-vector-transduced control fellow corneas. Epithelial wound healing time in c-met-transduced diabetic corneas decreased twofold compared with rAV-vector-transduced corneas and became similar to normal. c-Met action apparently involved increased activation of p38 mitogen-activated protein kinase. c-Met transduction did not change tight junction protein patterns, suggesting unaltered epithelial barrier function. Conclusions. rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. c-Met gene therapy could be useful for correcting human diabetic corneal abnormalities.

Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes

Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1–2 × 10 8 plaque forming units of rAV per cornea were added to cultures for 48 h with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6–10 days additional incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol. Sildenafil significantly increased epithelial transduction efficiency, apparently by stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin α 3β 1, nidogen-1, nidogen-2, and laminin γ2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic corneas. The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.

Bacterial Contamination of Human Organ-Cultured Corneas

This study was designed to define the risk of contamination of human corneas preserved by the organ-culture method. We examined the microbial contaminations in 3,100 corneoscleral rims cultivated in our eye bank. Microbiologic tests were performed in the preservation medium 5 days after the beginning of cornea cultures and in the last day of culture (21.5 +/- 8.1 days), when the corneas were transferred to the deswelling medium. In 1,029 corneas a microbiologic test also was performed 1 day after the beginning of deswelling procedure. We found 206 microbial contaminations (6.65% of total) after 5 days and 17 (0.55%) at the end of the preservation period. The total number of contaminated samples during the cornea culture was 223 corresponding to 7.2% of the samples (95% confidence interval, 6.3-8.1). The 1,029 tests performed during the deswelling step disclosed 26 contaminated cornea cultures despite apparent sterility of the medium (2.5%; 95% confidence interval, 1.5-3.5). The observation of microbial contaminations in a time close to the transplant (i.e., at the end of the preservation period and in the deswelling step) showed that a fast microbial tests during the deswelling procedure may prevent the grafting of a contaminated cornea. The appearance of bacteria in the deswelling medium despite a negative culture medium suggests that bacteria penetrate the corneal tissues during the culture to be subsequently extruded when the internal fluids move outward.

Bacterial contamination of human organ-cultured corneas.

The aim of this study was to detect and identify bacterial contaminants in human corneoscleral tissue after organ culture storage. Seventy-two corneoscleral rims and corneal buttons trephined from organ cultured corneoscleral discs using aseptic technique and 45 organ-cultured donor corneoscleral rims postpenetrating keratoplasty) were subjected to a mechanical extraction technique using a Stomacher laboratory blender. As a control, 28 of the corneoscleral rims and buttons were halved; one half of each corneoscleral rim and button was decontaminated in formalin for 48 hours before thorough washing in balanced salt solution. Corneal specimens, culture medium, and transport (5% dextran) medium were cultured in brain-heart infusion broth at 37 degrees C for 5 days. Bacterial isolates were identified after culture of turbid enrichment broth. Bacterial contamination was demonstrated in 29% (21 of 72) of the corneoscleral rims and 15% (11 of 72) of the corneal buttons that were trephined aseptically from corneoscleral discs and in 29% (13 of 45) of postkeratoplasty corneoscleral rims. Bacterial contaminants were not isolated from controls. Isolated microorganisms included coagulase-negative Staphylococcus, Staphylococcus aureus, Streptococcus viridans, Pseudomonas sp, and Bacillus sp. A correlation was not demonstrated between contamination and cause of death, death to enucleation, death to culture time, or time in culture. Postkeratoplasty endophthalmitis was not evident in the patients who had received corneal buttons from those corneoscleral discs that had contaminated corneoscleral rims. Bacterial contamination exists in corneoscleral tissue after organ culture storage. The difference in distribution of bacteria and percentage of contamination between the peripheral and central corneas causes us to question the value of routine postpenetrating keratoplasty corneoscleral rim cultures.