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