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