Evaluation of cardiac parameters and other safety outcomes of brolucizumab treatment in patients with neovascular age-related macular degeneration.

This was a prospective, single-dose, single-arm, open-label, non-randomized, multicenter clinical study to determine cardiovascular safety after a single brolucizumab 6 mg intravitreal injection in neovascular age-related macular degeneration patients (N = 14). Electrocardiogram (ECG) data were collected at different time points using 12-lead Holter and standard ECG, and patients were followed up to 8 days (end of study) for any signs of ocular and non-ocular adverse events (AEs). No clinically meaningful changes were observed in cardiac parameters. No patient had a ≥30 msec change from baseline in heart rate-corrected QT using Fridericia's formula (QTcF), and no patient had a new QTcF value of ≥450 msec between 20 and 24 h after treatment. No deaths or serious AEs were reported during the study period. These results are in line with the absence of new cardiovascular safety signal based on the ECG recordings collected over the first year of the pivotal studies performed with brolucizumab in DME. Trial Registration: ClinicalTrials.gov identifier: NCT03954626.

Pterygium Pathology: A Prospective Case-Control Study on Tear Film Cytokine Levels.

Pterygium is a common eye disease, linked to an increased exposure to UV radiation and dry environments. The associated pathology culminates in visual impairment and, in some rare cases, blindness. However, there remains a lot of uncertainty concerning the pathogenesis of this fibrovascular lesion. As the composition of the tear film provides a reflection into the pathological changes at the ocular surface, tear analysis represents an ideal approach to gain insight in the progression of disease following pterygiectomy. This study enrolled 19 patients and age/gender-matched healthy controls. Tear film levels of interleukin- (IL-) 6, IL-8, and vascular endothelial growth factor (VEGF) were investigated over time, and preoperative concentrations were linked to corneal neovascularization and pterygium size. Diminished tear film levels were found in unilateral patients who show no clinical signs of pterygium recurrence over a period of one year. Hence, our results highlight the potential of using the course of IL-6, IL-8, and VEGF levels in tears as biomarkers for recovery. In addition, when focusing on the affected eyes (i.e., primary and recurrent pterygium), we detected fold changes in preoperative cytokine concentrations to correspond with disease severity. As our proposed biomarkers did not reveal a linear relationship with corneal neovascularization nor the invasive behaviour of pterygium, no exact role in the pterygium pathology could be established. Hence, our data point to these factors being contributors rather than decisive players in the pathological processes.

Short- and Long-Term Results of Xenogeneic-Free Cultivated Autologous and Allogeneic Limbal Epithelial Stem Cell Transplantations.

PURPOSE: To evaluate the short- and long-term success rates of xenogeneic-free cultivated limbal epithelial stem cell transplantation (CLET) for the treatment of limbal stem cell deficiency (LSCD). METHODS: Thirteen patients with LSCD underwent an autologous (n = 9) or allogeneic (n = 4) CLET. The primary end point was to assess the long-term anatomical success rate of transplanted grafts at a follow-up of at least 3 years, in comparison with the short-term outcomes. Secondary end points involved reviewing functional improvement, patient-reported symptoms, and change in percentage area of corneal vascularization in both short-term and long-term. RESULTS: The mean short- and long-term follow-up periods were 2.1 ± 0.38 years and 6.7 ± 1.81 years, respectively. The total anatomical success rate was 46.1% in the short-term, but it decreased to 23.1% in the long-term. A partial success rate of 30.8% was observed in both short- and long-term, and the failure rate increased from 23.1% to 46.1%. The mean percentage of vessel area decreased from 12.11% ± 5.29% preoperatively to 7.82% ± 6.70% in the short-term and increased to 8.70% ± 6.32% in the long-term. There was a significant improvement in best-corrected visual acuity (P = 0.044) in the short-term although not in the long-term (P = 0.865). CONCLUSIONS: This study shows that anatomical and functional success rates of CLET decrease over time. We believe that the decline of success is related to the extent of disease, cell origin, and lack of niche protection because subtotal LSCD and autologous donor cells confer a higher chance of success in the long-term.

Optimized Protocol for Regeneration of the Conjunctival Epithelium Using the Cell Suspension Technique.

PURPOSE: To develop autologous tissue-engineered conjunctival epithelial sheets to be used as advanced therapy medicinal products for severe ocular surface disorders involving the conjunctiva. METHODS: Methods used aimed at 1) mapping the conjunctiva for identification of the stem cell location, 2) establishing proper cell culturing conditions, 3) identifying the proper scaffold, and 4) characterizing the conjunctival grafts better. For these purposes, immunostaining and PAS staining, serial cultivation of cells, and quantitative polymerase chain reaction ([INCREMENT]Np63α and MUC5AC) were performed. RESULTS: The inferior fornix represents the ideal area where to take the conjunctival biopsies from, with at least +3.58% of clonogenic colonies and higher percentages of stem cells compared with other areas, as confirmed by [INCREMENT]Np63α expression levels (6.79% ± 1.18%). The standard culture conditions are necessary when cells are cultured on bare plastic, while animal-free media can be used for conjunctival cell culture on the scaffold. Fibrin glue represents the ideal scaffold for production of epithelial conjunctival grafts because it allows physiological expression of the main conjunctival cell markers, with K19 as the ideal one (98.5% ± 0.5% positive cells). The presence of goblet cells (6.3% ± 1.3%) and expression of the stem cell marker [INCREMENT]Np63α (1.65% ± 0.35% positive cells) were also assessed. CONCLUSIONS: Our findings pave the way for ex vivo cultivation of conjunctival epithelial cells onto a scaffold using the cell suspension technique by means of animal-free media. This would allow us to obtain conjunctival grafts for clinical purposes, thus giving a therapeutic option to patients with conjunctival diseases refractory to current therapies.

Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants.

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. STATEMENT OF SIGNIFICANCE: Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

Modified bean-shaped ring segments for suture fixation of the bag-in-the-lens intraocular implant.

We describe a surgical technique for secondary stabilization of a bag-in-the-lens intraocular lens (BIL IOL) using 2 modified bean-shaped ring segments in cases of zonular dehiscence associated with pseudophakodonesis. The first modified bean segment is anchored in the sulcus with a suture to the sclera in the area of maximum zonular dehiscence, and the second segment is implanted in the opposite sulcus area. Both segments are placed in the BIL IOL interhaptic groove. The segments stabilize and center the BIL IOL by creating an artificial zonule that provides the necessary extra support for the IOL.

Optimisation of HPMC ophthalmic inserts with sustained release properties as a carrier for thermolabile therapeutics.

A methodology was developed and optimised for the preparation of a new drug delivery system (DDS) with sustained release properties to allow ocular protein delivery and to limit destructive production steps during manufacturing. Elevated temperatures, shear forces and an oxidative environment should be avoided in order to prevent denaturation or oxidation of proteins. An aqueous HPMC solution was prepared using heat and casted into small semi-rod-shaped PVC blisters. The polymer solution was allowed to cool down and was partially dehydrated at room temperature. A drug solution containing glycerol, drug and water was subsequently added to rehydrate the partially dehydrated polymer matrix at a temperature of 2°C. Several parameters of the production process were varied to determine their influence on the release kinetics from HPMC inserts from three different molecules of different molecular weight. This production method was further optimised in order to shorten the rehydration time from weeks to days, while eliminating heat and shear forces on the selected drug molecules sodium fluorescein, lysozyme and albumin. Slow release kinetics were achieved for sodium fluorescein and lysozyme as model drug molecules. The higher molecular weight of albumin prevented a good penetration into the insert during the rehydration process resulting in predominantly burst release. The biocompatibility of a viscous HPMC solution was evaluated on SV40-human corneal epithelial cells with PrestoBlue® and no cytotoxic effects were observed.

Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma.

Purpose: The human cornea has recently been described as a source of corneal stroma-derived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. Methods: This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers (CD73+, CD90+, CD105+, CD19-, CD34-, CD79α-, CD11b-, CD14-, CD45-, and HLA-DR-) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. Results: HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS- and HPL-supplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. Conclusions: We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs.

Validation of an Objective Keratoconus Detection System Implemented in a Scheimpflug Tomographer and Comparison With Other Methods.

PURPOSE: To validate a recently developed program for automatic and objective keratoconus detection (Keratoconus Assistant [KA]) by applying it to a new population and comparing it with other methods described in the literature. METHODS: KA uses machine learning and 25 Pentacam-derived parameters to classify eyes into subgroups, such as keratoconus, keratoconus suspect, postrefractive surgery, and normal eyes. To validate this program, it was applied to 131 eyes diagnosed separately by experienced corneal specialists from 2 different centers (Fondation Rothschild, Paris, and Antwerp University Hospital [UZA]). The agreement of the KA classification with 7 other indices from the literature was assessed using interrater reliability and confusion matrices. The agreement of the 2 clinical classifications was also assessed. RESULTS: For keratoconus, KA agreed in 92.6% of cases with the clinical diagnosis by UZA and in 98.0% of cases with the diagnosis by Rothschild. In keratoconus suspect and forme fruste detection, KA agreed in 65.2% (UZA) and 100% (Rothschild) of cases with the clinical assessments. This corresponds with a moderate agreement with a clinical assessment (κ = 0.594 and κ = 0.563 for Rothschild and UZA, respectively). The agreement with the other classification methods ranged from moderate (κ = 0.432; Score) to low (κ = 0.158; KISA%). Both clinical assessments agreed substantially (κ = 0.759) with each other. CONCLUSIONS: KA is effective at detecting early keratoconus and agrees with trained clinical judgment. As keratoconus detection depends on the method used, we recommend using multiple methods side by side.

A method for quantifying limbal stem cell niches using OCT imaging.

AIMS: To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings. METHODS: The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures. RESULTS: FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (ΔPR) and the mean interpalisade epithelial rete peg width (ΔERP) were both of the order of 72 µm, leading to a palisade density (PD) of about 7.4 palisades/mm. A significant difference in ΔPR, ΔERP and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters ΔPR, ΔERP and PD was found, and of age on PD and ΔERP (p<0.05). CONCLUSIONS: In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.

Mounting of Biomaterials for Use in Ophthalmic Cell Therapies.

When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for biomaterials is therefore a significant and common problem. In this review, we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance, and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of 4 experimental designs developed within our respective laboratories in Australia, the United Kingdom, and Belgium.

Evaluation of a Machine-Learning Classifier for Keratoconus Detection Based on Scheimpflug Tomography.

PURPOSE: To evaluate the performance of a support vector machine algorithm that automatically and objectively identifies corneal patterns based on a combination of 22 parameters obtained from Pentacam measurements and to compare this method with other known keratoconus (KC) classification methods. METHODS: Pentacam data from 860 eyes were included in the study and divided into 5 groups: 454 KC, 67 forme fruste (FF), 28 astigmatic, 117 after refractive surgery (PR), and 194 normal eyes (N). Twenty-two parameters were used for classification using a support vector machine algorithm developed in Weka, a machine-learning computer software. The cross-validation accuracy for 3 different classification tasks (KC vs. N, FF vs. N and all 5 groups) was calculated and compared with other known classification methods. RESULTS: The accuracy achieved in the KC versus N discrimination task was 98.9%, with 99.1% sensitivity and 98.5% specificity for KC detection. The accuracy in the FF versus N task was 93.1%, with 79.1% sensitivity and 97.9% specificity for the FF discrimination. Finally, for the 5-groups classification, the accuracy was 88.8%, with a weighted average sensitivity of 89.0% and specificity of 95.2%. CONCLUSIONS: Despite using the strictest definition for FF KC, the present study obtained comparable or better results than the single-parameter methods and indices reported in the literature. In some cases, direct comparisons with the literature were not possible because of differences in the compositions and definitions of the study groups, especially the FF KC.

Optimization and validation of an existing, surgical and robust dry eye rat model for the evaluation of therapeutic compounds.

The aim of this research was to optimize and validate an animal model for dry eye, adopting clinically relevant evaluation parameters. Dry eye was induced in female Wistar rats by surgical removal of the exorbital lacrimal gland. The clinical manifestations of dry eye were evaluated by tear volume measurements, corneal fluorescein staining, cytokine measurements in tear fluid, MMP-9 mRNA expression and CD3(+) cell infiltration in the conjunctiva. The animal model was validated by treatment with Restasis(®) (4 weeks) and commercial dexamethasone eye drops (2 weeks). Removal of the exorbital lacrimal gland resulted in 50% decrease in tear volume and a gradual increase in corneal fluorescein staining. Elevated levels of TNF-α and IL-1α have been registered in tear fluid together with an increase in CD3(+) cells in the palpebral conjunctiva when compared to control animals. Additionally, an increase in MMP-9 mRNA expression was recorded in conjunctival tissue. Reference treatment with Restasis(®) and dexamethasone eye drops had a positive effect on all evaluation parameters, except on tear volume. This rat dry eye model was validated extensively and judged appropriate for the evaluation of novel compounds and therapeutic preparations for dry eye disease.

Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies.

Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.

Evaluation of UVA Cytotoxicity for Human Endothelium in an Ex Vivo Corneal Cross-linking Experimental Setting.

PURPOSE: To evaluate endothelial cytotoxicity after exposure of human corneas to ultraviolet-A (UVA) (λ = 365 nm; 5.4 J/cm(2)) in an experimental ex vivo corneal cross-linking setting. METHODS: Sixteen pairs of human donor corneas were cut into two pieces. One piece of each cornea was treated with 0.025% riboflavin solution prior to UVA irradiation (5 minutes; 18 mW/cm(2)), whereas the other piece was not irradiated but treated with riboflavin (right eye) or preservation medium (left eye). By irradiating from the endothelial side, the UVA dosage applied to endothelial cells exceeded at least eight times the cytotoxic threshold established in animal models (0.65 J/cm(2)). Endothelial cell counts were performed by two independent investigators after storage (4 to 5 days at 31 °C) and staining (trypan blue, alizarin red). Normality (Q-Q plot; Shapiro-Wilk test) and equivalence (mixed-effects modeling with a 10% equivalence threshold) of the endothelial cell counts of the different groups were evaluated. RESULTS: Equivalence of mean endothelial cell density between both groups was observed: 2,237 ± 208 cells/mm(2) in UVA-irradiated pieces and 2,290 ± 281 cells/mm(2) in control pieces (mean difference of 53 ± 240 cells/mm(2) between both groups). CONCLUSIONS: Despite direct irradiation of human donor endothelium using the clinical dosage for cross-linking, equivalence in endothelial cell counts was observed between irradiated tissues and controls. Ex vivo human corneal endothelial cells seem to be far more resistant to riboflavin-enhanced UVA irradiation than previously estimated by animal experiments.

Optical Coherence Tomography in Cultivated Limbal Epithelial Stem Cell Transplantation Surgery.

PURPOSE: Cultivated limbal epithelial stem cell transplantation (CLET) is a surgical approach reserved for severe stem cell deficiency and anterior segment pathology. The recipient cornea typically displays a highly vascularized pannus that must be dissected before graft placement. Extensive pannus dissection, however, can result in corneal thinning and increase the risk of ectasia and ultimately, of perforation. We aimed to examine the value of anterior segment optical coherence tomography (AS-OCT) intraoperatively during pannus resection and long-term postoperative follow-up of CLET surgery. DESIGN: This was a prospective observational case series. METHODS: This study was performed on 8 eyes of 8 patients who underwent CLET surgery. Intraoperative OCT was performed before dissection and at several time points during dissection and after placement of the ex vivo cultivated graft. During the postoperative follow-up period, OCT was performed to image the graft integration and corneal thickness. RESULTS: Anterior segment optical coherence tomography provided high-resolution images of the corneal pannus, including the blood vessel lumen. Imaging of the pannus depth and cleavage plane was possible intraoperatively, with the patient supine. Postoperative imaging displayed the transplanted graft, temporary amniotic membrane patch, and overlying bandage contact lens. The grafts were also visible after removal of the temporary membrane patch. Four months postoperatively, the grafts were integrated into the cornea, and all 8 participants showed the presence of an epithelial layer. CONCLUSIONS: During CLET surgery, AS-OCT offers a noncontact method for rapid, intraoperative scanning and pachymetry mapping. Intraoperative OCT guidance assists in the removal of the delicate fibrovascular pannus. Sparing the viable corneal stroma can reduce the risk of extreme thinning, iatrogenic ectasia, and wound dehiscence while providing a smooth recipient surface for the composite graft.

Intraocular lens exchange technique for an opacified bag-in-the-lens.

We describe the intraocular lens (IOL) exchange technique that is specific to the bag-in-the-lens IOL. The subsequent IOL analysis displayed a deep granular opacification consisting predominantly of calcium and phosphates that has been described in hydrophilic IOLs.

Repeatability and inter-device agreement for three different methods of keratometry: Placido, Scheimpflug, and color LED corneal topography.

PURPOSE: To determine the repeatability of a color LED corneal topographer (Cassini; iOptics, The Hague, The Netherlands) and compare it with Placido and Scheimpflug based devices (EyeSys 2000; EyeSys Laboratories, Houston, TX, and Pentacam HR; Oculus Optikgeräte GmbH, Wetzlar, Germany). METHODS: This prospective study involved 20 healthy volunteers (20 eyes) recruited from the staff of the Antwerp University Hospital. For each eye, three measurements were taken using each device, from which eight parameters describing keratometry and astigmatism were derived. Repeatability was assessed using intrasession, within-subject analysis of variance. Agreement was evaluated between pairs of devices with Bland- Altman plots and 95% confidence intervals. RESULTS: The repeatability values were good for all three devices, although slightly worse for the Cassini than for the other two devices for steep, flat, and mean keratometry. The EyeSys showed worse repeatability for the astigmatism axis. EyeSys and Pentacam agreed well with each other in terms of mean keratometry, whereas the Cassini gave consistently higher values by 0.52 diopters (D) when compared with the EyeSys (P < .05, paired t test) and by 0.38 D when compared with the Pentacam (P < .05, paired t test). The Cassini provided similar repeatability values (2.31°) for the astigmatism axis to the Pentacam (2.22°), in contrast to the EyeSys, which produced much more variable axes (9.0°) (P < .05, paired t test). CONCLUSIONS: Overall, the three devices display comparable repeatability. The one exception is the astigmatism axis measurement of the EyeSys. The Cassini provides higher keratometry values than the other two devices, but the astigmatism axis agrees well with that provided by the Pentacam.

Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation.

BACKGROUND: To determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency. METHODS: In vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique. RESULTS: The majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity. CONCLUSION: We conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

Lymphangiogenesis may play a role in cultivated limbal stem cell transplant rejection.

We describe the clinicopathologic findings in a case of acute rejection following cultivated limbal stem cell transplantation (CLSC). This is the first time lymphangiogenesis has been described in CLSC rejection. This indicates that 1. absence of corneal stromal cells or 2. down regulation of HLA antigens by exposure to culture conditions, does not reduce the risk of graft rejection when transplanting into a high risk corneal bed. Our findings highlight the need for development of anti-lymphangiogenic treatments prior to CLSC transplantation.