Lipofuscin granules from retinal pigment epithelium (RPE) cells contain bisretinoid fluorophores, which are photosensitizers and are phototoxic to cells. In the presence of oxygen, bisretinoids are oxidized to form various products, containing aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that bisretinoid oxidation and degradation products have both hydrophilic and amphiphilic properties, allowing their diffusion through the lipofuscin granule membrane into the RPE cell cytoplasm, and are thiobarbituric acid (TBA)-active. The purpose of the present study was to determine if these products exhibit a toxic effect to the RPE cell also in the absence of light. The experiments were performed using the lipofuscin-fed ARPE-19 cell culture. The RPE cell viability analysis was performed with the use of flow cytofluorimetry and laser scanning confocal microscopy. The results obtained indicated that the cell viability of the lipofuscin-fed ARPE-19 sample was clearly reduced not immediately after visible light irradiation for 18 h, but after 4 days maintaining in the dark. Consequently, we could conclude that bisretinoid oxidation products have a damaging effect on the RPE cell in the dark and can be considered as an aggravating factor in age-related macular degeneration progression.

Background: Limbal stem cell deficiency (LCSD) presents several challenges. Currently, there is no clearly defined systematic approach to LSCD diagnosis that may guide surgical tactics. Methods: The medical records of 34 patients with LSCD were analyzed. Diagnostic modalities included standard (visometry, tonometry, visual field testing, slit-lamp biomicroscopy with corneal fluorescein staining, Schirmer test 1, ultrasonography) and advanced ophthalmic examination methods such as anterior segment optical coherence tomography, in vivo confocal microscopy, impression cytology, and enzyme-linked immunoassay. Results: Standard ophthalmological examination was sufficient to establish the diagnosis of LSCD in 20 (58.8%) cases, whereas advanced evaluation was needed in 14 (41.2%) cases. Depending on the results, patients with unilateral LSCD were scheduled to undergo glueless simple limbal epithelial transplantation (G-SLET) or simultaneous G-SLET and lamellar keratoplasty. Patients with bilateral LSCD with normal or increased corneal thickness were enrolled in the paralimbal oral mucosa epithelium transplantation (pLOMET) clinical trial. Conclusions: Based on the diagnostic and surgical data analyzed, the key points in LSCD diagnosis were identified, helping to guide the surgeon in selecting the appropriate surgical procedure. Finally, we proposed a novel step-by-step diagnostic algorithm and original surgical guidelines for the treatment of patients with LSCD.

Relevance. In recent years, due to the shortage of donor corneas, the need to creating conditions for storing stromal lenticules in eye banks for their clinical use in ophthalmosurgery have been actively discussed. Currently, scientists are looking for optimal conditions for storage native lenticules. There were no reports of storage of decellularized lenticules in the literature. Purpose. To develop optimal conditions for cryopreservation of stromal tissue-engineered constructs for the subsequent creation of a cryobank. Material and methods. The optical properties of native lenticules and tissue–engineered corneal constructs (TCs) were assessed using spectrophotometry. We used a decellularization protocol with 1.5 M NaCl with DNase 5 U/ml and RNase 5 U/ml to create TC. Dispersed viscoelastic agent approved for clinical use in ophthalmology was used for dehydration of TC. Three comparison groups were formed: 1st – control group (native lenticules), 2nd – group without dehydration of TC, 3rd – group with dehydration of TC. The spectrophotometer data was evaluated in 2 stages. The transparency of the control group was measured at 1 stage. At the second stage, the transparency of two experimental groups after storage in DMSO was investigated (a group without dehydration of TC and a group with dehydration of TC). Results. When compared between groups without dehydration of TC; with dehydration of TC and the control group (p≥0.05), no statistical difference was revealed, and when comparing groups without dehydration of TC and groups with dehydration of TC (p≥0.05), no statistical difference was revealed. Conclusion. Groups with dehydration of TC and without dehydration of TC after storage in DMSO did not differ in transparency. In this regard, these groups should be considered as interchangeable in terms of optical properties. Key words: storage, cryopreservation, lenticule, cornea, decellularization, tissue engineering

Background.The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions.Aims to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering.Methods.Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patients lip mucosa were used to set the explant (n=18) and enzymatic (n=17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06mM Calcium contained 5% fetal bovine serum, 5 g/ml human insulin, 5 g/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression.Results.The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p=0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.13.6 m thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p=0.001) in comparison with the enzymatic method. Stable cultures had a cobble-stone morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mMCa++medium (p=0.0006).Conclusions.Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.

BACKGROUND: Investigation of morphological changes in the trabecular meshwork after manual trabeculotomy may support the hypotensive effect of this procedure. AIM: The study aimed to assess changes in the trabecular meshwork of the anterior chamber angle after experimental modeling of ex vivo manual trabeculotomy. METHODS: After experimental modeling of ex vivo manual trabeculotomy, morphology of the trabecular meshwork of 4 cadaver eyes was studied using scanning electron microscopy, and aqueous outflow was visualized in 4 cadaver eyes using an injection technique. RESULTS: Analysis of scanning electron microscopy results showed that manual trabeculotomy leads to partial shedding of the uveal meshwork, leaving corneal endothelium completely intact. The injection technique demonstrated that the dispersed dye is found at all levels of trabecular outflow in the area of trabecular meshwork scraping compared with the intact area, where the dye spread is limited by the trabecular meshwork and Schlemm canal. CONCLUSION: This experimental study of the trabecular meshwork after manual trabeculotomy suggests a possible improvement in aqueous outflow.