Первый опыт трансплантации 3D-сфероидов ретинального пигментного эпителия в эксперименте

Abstract

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.