Survival of the posterior lamellar cornea graft keratocytes and endothelial cells cultivated in the modified corneal preservation media

Purpose. To study the survival of keratocytes and endothelial cells of a human donor cornea storage in the standard and the new media which was specifically designed for optim ized cornea hydration.Material and methods. 2D cell cultures of keratocytes and endothelial cells obtained from the Eye tissue bank were used for culture in improved storage media over a period of 14 and 7 days subsequently. To confirm phenotype characteristics, the cells were stained by the following markers: for keratocytes – Lumikan, Keratocan, and α-smooth muscle actin; for endothelial cells – ZO-1 and Na/K-ATPase. The onset of apoptosis in cell culture of keratocytes were detected with Cytochrome C, BAX, and Caspase 3 and 8. Viability of cell cultures after the cultivation was carried out using a commercial set of «Live and Dead». Morphology of the endothelial cells was assessed using an electron scanning microscope.Results. It was shown that the 2D keratocyte culture cultured in the improved storage media expressed specific markers: Lumican, Keratocan, and did not express α-smooth muscle actin. There were no markers of apoptosis in the cell culture of keratocytes after 14 days of cultivation. Corneal endothelium cultured in the improved storage media expresses  ZO-1, Na/K-ATPase and presented hexagonal cell shape morphology according to electron microscopy.Conclusion. The improved storage media allow to preserve the unique phenotype of keratocytes, with a slight decrease in proliferative cells activity during 14 days. The media maintain a viable and functional corneal endothelium for at least seven da ys of cultivation

PREOPERATIVE PREPARATION OF LIMBAL CELL TRANSPLANTS FOR THE TREATMENT OF OPTIC NEUROPATHY (EXPERIMENTAL STUDY)

Purpose. To study experimentally in vitro secretion of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF) using intact and induced multi-potent mesenchymal limbal stem cells (MSCs) in three-dimensional culture (3D).Material and methods. MSCs were obtained by culturing of limbal fragments, isolated from the cadaveric human donor eyes, according to the medical technology of the S. Fyodorov Eye Microsurgery Federal State Institution. The phenotype of obtained cell culture was studied by the flow cytometry method.Stimulation of secretion of neurotrophic factors was performed via a two-step technique using non-specific activation factors: EGF, hbFGF, N2 additive, dibutyryl cAMP, NRG1-beta 1, PDGF, 3-isobutyl-1- methylxanthine. The 3D-cell spheroids were generated using agarose plates (3D Petri dishes, Microtissue, USA) for three comparative groups where: Group I – control, Group II – with the induction of spheroids at 1 day of cultivation; Group III – with induction of spheroids at 7 days of cultivation. The concentration of NGF and BDNF in the culture medium was studied using the enzyme linked immunosorbent assay (ELISA).Results. The induction of the 3D spheroids of limbal MSCs, that carried out on the 1st and 7th days of incubation, contributes to a significant increase in the production of NGF and BDNF, but subsequently a pronounced reduction in the secretion of these factors is observed. The conducting of an induction leads to a change in the morphology of spheroids: loss of compactness, the emergence of «fringed» (debris). Such changes indicate a non-viability of the obtained 3D-cell spheroids.Conclusion. The cellular spheroids, created from 2D-culture of intact limbal MSCs by the three-dimensional culture method, are capable in sufficient therapeutic concentrations spontaneously to synthesize NGF and BDNF, have the most optimal design for transplantation in extrabulbar and intraocular tissue niches of the eyeball, are a potential source of prolonged secretion of neural basis function in cell treatment of optic neuropathy

Разработка техники трансплантации 3D-клетонных сфероидов ретинального пигментного эпителия в опыте на животных

Aim. This research is aimed to devise the technique for transplantation of 3D spheroids retinal pigment epithelium (RPE) in the experimental animal’s eyes (rabbits).Materials and methods. 3D spheroids of RPE for subsequent transplantation were created using agarose tablets (3D Petri Dishes, Microtissue, USA). The phenotype of the obtained cell cultures was studied by immunocytochemical tests (laser scanning confocal microscope “Fluo View FV10i”, Olympus, Japan). Vitrectomy - 2500 cuts per minute, vacuum 600 mm Hg (Alcon, Accurus, USA) was performed on all rabbits (n = 10). Then, we made retinotomy and injected spheroids in subretinal space (MicroDose injection kit 1 ml, Med One, USA). The following methods of control: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT), (Askin Spectralis, Heidelberg engineering, Germany). Eyes were enucleated for histological examination on 7, 10, 14 and 20 days.Results. Immunocytochemical tests revealed preservation of the RPE epithelial phenotype in 3D spheroids. Clinical map was similar in all experimental animals - during the first 7 days after surgery we saw cystic edema and flat retinal detachment in the surgery area. As we observed, the retina was adjoining and retinal edema was decreasing. Also, on day 3, 7 and 10 on OCT we saw subretinal round conglomerates with a diameter of 60 to 80 µm - presumably RPE 3D spheroids. According to histological findings, there was observed adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of new cell layer with the increase of observation periods.Conclusion. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells. The developed surgical technique of RPE transplantation is acceptable and can be used for further experimental studies to be implemented in clinical practice.Цель исследования. Разработка техники трансплантации SD-клеточных сфероидов ретинального пигментного эпителия (РПЭ) на глазах экспериментальных животных (кролики).Материалы и методы. SD-сфероиды РПЭ для последующей трансплантации создавали с использованием агарозных планшетов (3D Petri Dishes, Microtissue, США). Фенотип полученных клеточных культур исследовали с помощью иммуноцитохимического анализа (лазерный сканирующий конфокальный микроскоп «Fluo View FV10i», Olympus, Япония). Всем экспериментальным животным (кролики породы шиншилла, n = 10) выполняли витрэктомию - 2500 резов в минуту, вакуум 600 мм рт. ст. (Alcon, Accurus, США), ретинотомию и субретинально вводили сфероиды РПЭ (MicroDose injection kit 1 ml, Med One, США). Методы послеоперационного контроля: ультразвуковое В-сканирование глаза (Ultrasonic UD-6000, Tomey, Япония) и оптическая когерентная томография - ОКТ (Askin Spectralis, Heidelberg engineering, Германия). Глазные яблоки энуклеировали на 7, 10, 14, 20-е сутки для последующего гистологического исследования.Результаты. Иммуноцитохимическое окрашивание выявило сохранение фенотипа РПЭ в форме 3D-сфе-роидов. В послеоперационном периоде у всех экспериментальных животных по данным ультразвукового В-сканирования и ОКТ отмечалась схожая клиническая картина: отек и плоская отслойка сетчатки в зоне оперативного вмешательства. По мере наблюдения сетчатка прилегала и отек сетчатки уменьшался. Также, по данным ОКТ, субретинально обнаруживались округлые конгломераты диаметром от 60 до 80 мкм - предположительно 3D-сфероиды РПЭ. По данным гистологического исследования отмечалась адгезия сфероидов РПЭ к сосудистой оболочке с последующим распластыванием и образованием нового клеточного слоя по мере увеличения сроков наблюдения.Заключение. Предложенная технология культивирования кроличьего РПЭ с последующим конструированием 3D-сфероидов позволяет сохранить эпителиальный фенотип клеток. Разработанная хирургическая техника трансплантации РПЭ является приемлемой и может использоваться для дальнейших экспериментальных исследований с целью внедрения в клиническую практику

Neurotrophic factors and cell therapy in the treatment of glaucomatous optic neuropathy

At present it is accepted that glaucoma is a multi-factorial neurodegenerative disease, in which process there occur a death of retinal ganglion cells (RGC), progressive optic neuropathy and a visual field loss.In recent years, a number of large multicenter studies have convincingly shown that the effective reduction of intraocular pressure by medication and surgical methods does not guarantee a long-term stabilization of the glaucomatous process, and therefore a number of patients have a progression of the neurodegenerative process.This fact determines the necessity to search new ways of glaucoma optic neuropathy therapy, one of these can be the neuroprotection based on the methods of cell therapy.It is shown that the apoptosis is the primary mechanism of RGS death in glaucoma as in other neurodegenerative diseases. Currently found a large number of triggers of RGS apoptosis. One of the most important is the factor blocking axoplasmatic transport of neurotrophins.Neurotrophins are a family of structurally and functionally similar polypeptides that plays an important role in the differentiation, survival and regeneration of neurons.Numerous studies have shown that the neurotrophic factors in general, especially brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) significantly improves the RGC survival in experimental models of glaucoma.It is revealed that multipotent mesenchymal stromal cells (MMSC) can release a large number of bioactive factors, including neurotrophins, both in vivo, as well as in experiments in vitro.MMSC of eye limbus phenotypic match the bone marrowderived mesenchymal stromal cells. During MMSC culturing they secrete a wide variety of cytokines, interleukins, growth-factors. Therefore we consider the transplantation of allogenic limbus fragments as a candidate for cell therapy in the treatment of glaucomatous optic neuropathy

Screening of cadaver cornea donor for infections in the eye bank of the Fyodorov Eye Microsurgery Federal State Institution

Objective: to analyze negative laboratory results of cadaver cornea donor screening during preparation of corneas for transplantation according to data from the internal registry of donors of the eye bank (EB) of the Fyodorov Eye Microsurgery Federal State Institution and the European Eye Bank Association (EEBA) from 2011 through 2015. Materials and methods. Data analysis was carried out using the internal registry of EB donors and the EEBA annual directories. The analyzed data included the number of eyeballs obtained, the frequency of incomplete tests (hemolysis for EB) and positive serological results for human immunodeficiency virus (HIV-1 and HIV-2), viral hepatitis B, viral hepatitis C and syphilis. Results. In just 5 years, the EB received 3,479 eyeballs. After hemolysis of donor blood samples, 13.9% (n = 486) of corneas were excluded from the EB. EEBA recorded fewer inconclusive tests during the same period. After hemolysis and positive serological tests, 19.4% (n = 676) of corneas were excluded from the EB. Overall, the number of positive serological tests in EBs was far higher than in the EEBA data. Frequency of positive HIV tests (HIV-1 and HIV-2) and syphilis in EB showed low variability annually, while incidence of hepatitis B increased in 2015. For the analyzed period, positive serology for hepatitis C was found to be prevalent among EB donors. Mixed infections were quite often recorded in blood samples. Conclusion. Based on analysis conducted, positive serology and hemolysis were the main contraindications and led to exclusion of 33.3% (n = 1162) of cadaver donor corneas received in EB. Frequency of positive serological tests for indicated infections in EB was higher than in the EEBA data, with significant predomination of hepatitis C

Development optimal conditions for cryopreservation of tissue-engineered corneal constructs

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

The development of transplantation technique of 3D spheroids retinal pigment epithelium in the experiment on animals

Aim. This research is aimed to devise the technique for transplantation of 3D spheroids retinal pigment epithelium (RPE) in the experimental animal’s eyes (rabbits).Materials and methods. 3D spheroids of RPE for subsequent transplantation were created using agarose tablets (3D Petri Dishes, Microtissue, USA). The phenotype of the obtained cell cultures was studied by immunocytochemical tests (laser scanning confocal microscope “Fluo View FV10i”, Olympus, Japan). Vitrectomy - 2500 cuts per minute, vacuum 600 mm Hg (Alcon, Accurus, USA) was performed on all rabbits (n = 10). Then, we made retinotomy and injected spheroids in subretinal space (MicroDose injection kit 1 ml, Med One, USA). The following methods of control: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT), (Askin Spectralis, Heidelberg engineering, Germany). Eyes were enucleated for histological examination on 7, 10, 14 and 20 days.Results. Immunocytochemical tests revealed preservation of the RPE epithelial phenotype in 3D spheroids. Clinical map was similar in all experimental animals - during the first 7 days after surgery we saw cystic edema and flat retinal detachment in the surgery area. As we observed, the retina was adjoining and retinal edema was decreasing. Also, on day 3, 7 and 10 on OCT we saw subretinal round conglomerates with a diameter of 60 to 80 µm - presumably RPE 3D spheroids. According to histological findings, there was observed adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of new cell layer with the increase of observation periods.Conclusion. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells. The developed surgical technique of RPE transplantation is acceptable and can be used for further experimental studies to be implemented in clinical practice

The first experience of 3D spheroids retinal pigment epithelium transplantation in the experiment

Introduction. Available methods in the treatment of age-related macular degeneration (AMD) do not always lead to significant vision improvement.A new advanced method of AMD treatment is transplantation of retinal pigment epithelium (RPE) in the form of cell suspension or choroidal pigment complex.In our opinion, the most modern form of RPE transplant is a multicellular spheroid - the form of 3D cell culture in which cells are close to the conditions of native tissue.However, transplantation of 3D spheroids of RPE requires preclinical studies.Purpose. This research is aimed to devise the technique for transplantation of RPE 3D spheroids in the eyes of experimental animals (rabbits).Material and methods. 1. In vitro research phase. For immunocytochemical tests the 3D spheroids were explored on the 3rd, 7th, and 11th day of steroidogenesis (using the laser scanning confocal microscope «Fluo View FV10i», Olympus, Japan). The expression of epithelial markers (Alexa Fluor, Great Britain), such as: RPE65, ZO-1, Cytokeratin 8, 18, and Vimentin (the mesenchymal marker) was analyzed.2. In vivo research phase. Vitrectomy (2500 cuts per minute, vacuum 600 mmHg), (Alcon, Accurus, USA) was performed on all rabbits (n=10). Then, a sharp cannula 39G was used to make a retinotomy above the central zone of retina, and spheroids (n=81) were injected (MicroDose injection kit 1 ml, Med One, USA) in subretinal space. The operation ended with the replacement of fluid into air and suturing scleral incision and the conjunctiva. The following methods of control were used: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT) - (Askin Spectralis, Heidelberg engineering, Germany).Animals were taken out of the experiment on days 7, 10, 14 and 20 by air embolism. The eyeballs were enucleated for a subsequent histological examination.Results. 1. In vitro research phase. During immunocytochemical tests on the obtained 3D cultures, the presence of high expression of specific marker of retinal pigment epithelium RPE-65, also epithelial markers Cytokeratin 8, 18 and ZO-1 was noted. The expression of mesenchymal marker Vimentin was weak - that indicates the advantage of 3D cultivation of RPE cells to keep their phenotype. This indicates the advantage of 3D cultivation of epithelial cells to preserve their epithelial phenotype.2. In vivo research phase. On day 1 during ultrasonic B-scanning in 6 rabbits there was observed a flat retinal detachment in the area of surgical intervention height up to 1 mm; in 4 rabbits there was detected adhesion of the membranes, detachment of retina was not visible.The picture of the morphological state during retinal OCT was similar in all experimental animals - during the first 7 days after surgery cystic edema was noted and also a flat retinal detachment in the surgery area. As we observed, the retina was attaching and retinal edema was decreasing. Also, on day 3, 7 and 10 we revealed subretinal round conglomerates with a diameter of 60 to 80 pm - presumably RPE 3D spheroids. No morphological changes of the retina were seen on day 14 and day 20.According to histological findings, there was found adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of a new cell layer with an increase of follow-up periods.Conclusion. 1. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells, that is confirmed by immunocytochemical tests.2. The developed surgical technique of RPE transplantation is acceptable, that is confirmed by the OCT and histological investigation.3. The proposed surgical technique of subretinal transplantation of 3D spheroids of RPE is promising for further experimental studies to be implemented in clinical practice

Сравнительный анализ протоколов децеллюляризации лентикулярной ткани роговицы

Shortage of donor corneas is a burning issue in ophthalmology. That is why there is a search for new alternative ways for treating corneal diseases. Decellularization technologies make it possible to create corneal tissue-engineered constructs that can adrress the issue of donor corneal shortage. Objective: to conduct a comparative analysis of effective methods for treating the corneal lenticula and to create an optimized and standardized decellularization protocol. Materials and methods. Corneal stromal lenticules obtained after ReLEx SMILE surgery were chosen for the study. Lenticule parameters: thickness 77–120 microns, diameter 6.5 mm. We used 3 protocols for the treatment of lenticules: 1) treatment with 1.5 M sodium chloride with nucleases (NaCl); 2) 0.1% SDS (SDS); 3) treatment with Trypsin-EDTA solution, followed by double washing in a hypotonic Tris buffer solution with nucleases (Trypsin-EDTA). Optical properties of lenticles were determined spectrophotometrically, where the samples before decellularization served as a control. Fluorescence imaging of nuclear material in the original cryosections was performed using Hoechst dye. The state of collagen fiber ultrastructure was assessed by scanning electron microscopy. The quantitative DNA content in fresh lenticules and in lenticules after treatment was analyzed. Results. All three decellularization protocols effectively removed nuclear and cellular material; the residual DNA content was < 50 ng/mg. However, the Trypsin-EDTA protocol led to significant damage to the extracellular matrix structure, which negatively affected the transparency of corneal tissue-engineered constructs. Transparency of samples for the NaCl protocol was close to native lenticules. Conclusion. To create a corneal tissue-engineered construct, NaCl decellularization protocols appear to be optimized and can be used to treat various corneal diseases.Введение. Актуальной проблемой офтальмологии является дефицит донорских роговиц. Данный факт обусловливает поиск новых альтернативных путей для лечения патологий роговицы. Технологии децеллюляризации позволяют создавать роговичные тканеинженерные конструкции, которые могут решить проблему нехватки донорских роговиц.Цель. Провести сравнительный анализ эффективных методов обработки роговичной лентикулы и создать оптимизированный и стандартизированный протокол децеллюляризации.Материалы и методы. Для исследования были выбраны стромальные роговичные лентикулы, полученные после операции ReLEx SMILE. Параметры лентикул: толщина 77–120 мкм, диаметр 6,5 мм. Для обработки лентикулы использовали 3 протокола: 1) обработка 1,5 М хлоридом натрия с нуклеазами (NaCl); 2) 0,1% SDS (SDS); 3) обработка раствором Трипсин-ЭДТА с последующем двойным отмыванием в гипотоническом трис-буферном растворе с нуклеазами (Трипсин-ЭДТА). Оптические свойства лентикул определяли спектрофотометрически, где в качестве контроля служили образцы до децеллюляризации. Определение структуры стромы роговичной лентикулы после децеллюляризации происходило с помощью окрашивания гематоксилином и эозином, по Ван-Гизону и альциановым синим. Также в качестве дополнительного метода оценки состояния внеклеточного матрикса, а именно коллагена I, III, V и VI типов, проводили иммуногистохимический анализ криосрезов нативных обработанных лентикул. Флуоресцентная визуализация ядерного материла в исходных криосрезах производилась с помощью красителя Hoechst. Состояние ультраструктуры коллагеновых волокон оценивалось с помощью сканирующего электронного микроскопирования. Производили анализ количественного содержания ДНК в свежих лентикулах и в лентикулах после обработки.Результаты. Все три протокола децеллюляризации эффективно удаляют ядерный и клеточный материал, остаточное содержание ДНК было < 50 нг/мг. Однако протокол с Трипсин-ЭДТА приводит к значительному повреждению структуры внеклеточного матрикса, что отрицательно сказывается на прозрачности роговичных тканеинженерных конструкций. Прозрачность образцов для протокола NaCl была приближена к нативным лентикулам.Заключение. Для создания роговичной тканеинженерной конструкции протокол децеллюляризации NaCl представляется оптимизированным и может применяться для лечения различных патологий роговицы