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.

1. Balashova LM. Vitaminno-mineral’nye kompleksy i zdorov’e glaz. Klinicheskaya oftal’mologiya. 2008; 9 (1): 41-45.

2. Bikbov MM, Fajzrahmanov RR. Operativnoe lechenie pacientov s fibrovaskulyarnymi membranami pri makulyarnoj degeneracii s chastichnym vosstanovleniem pig-mentnogo epiteliya setchatki. Sovremennye tekhnologii v oftal’mologii. 2017; 1: 35-38.

3. Borzenok SA, Kosheleva AV, Popov IA, Saburina IN i dr. 3D-kul’tivirovanie retinal’nogo pig-mentnogo epiteliya trupnyh donorskih glaz chelove-ka na predtransplantacionnom etape. VII Vserossijskij s»ezd transplantologov. Materialy s»ezda. M., 2014: 262-263.

4. Libman ЕS, Shahova Е1. Slepota, slabovidenie i invalidnost’ po zre-niyu v Rossijskoj Federacii. Rossijskij mezhregional’nyj simpozium «Likvidaciya ustranimoj slepoty: Vsemirnaya iniciativa VOZ»: Materialy. M., 2003: 38-42.

5. Algvere PV Berglin L, Gouras P, Sheng Y, Kopp ED. Transplantation of RPE in agtrelated macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch Clin Exp Ophthalmol. 1997; 235 (3): 149-158.

6. Binder S, Krebs I, Hilgers RD, Abri A, Stolba U, Assa-doulina A et al. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest Ophthalmol Vis Sci. 2004; 45 (11): 4151-4160.

7. Binder S, Stolba U, Krebs I, Kellner L, Jahn C, Feichtinger H et al. Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: a pilot study. Am J Ophthalmol. 2002; 133 (2): 215-225.

8. Bindewald A, Roth F, Van Meurs J, Holz FG. Transplantation of retinal pigment pithelium (RPE) following CNV removal in patients with AMD. Techniques, results, outlook. Ophthalmologie. 2004; 101 (9): 886-894.

9. Caramoy A, Liakopoulos S, Kirchhof B. Recurrence of choroidal neovascular membrane after autologous transplantation of RPE and choroid for neovascular AMD. Acta Ophthalmol. 2011; 89 (8): 666-668.

10. Falkner-Radler CI, Krebs I, Glittenberg K et al. Human retinal pigment epithelium (RPE) transplantation: outcome after autologous RPE-choroid sheet and RPE cell-suspension in a randomized clinical study. Br. J. Ophthalmol. 2011; 95: 370-375.

11. Cuiping Zhao, Nathan C. Boles, Justine D. Miller, Suzanne Kawola, Sally Temple, Richard J. Davis, Jeffrey H. Stern Development of a Refined protocol for trans - scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes. Journal of Visualized Experiments. (126), e55220.

12. Del Priore LV, Kaplan HJ, Tezel TH, Hayashi N, Berger AS, Green WR. Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation. Am J Ophthalmol. 2001; 131 (4): 472-480.

13. Eyetech Study Group. Antivascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to agerelated macular degeneration: phase II study results. Ophthalmology. 2003; 110 (5): 979-986.

14. van Meurs JC, Eter Averst, Hofland LJ, van Hagen PM, Mooy CM, Baarsma GS et al. Autologous peripheral retinal pigment epithelium translocation in patients with subfoveal neovascular membranes. Br J Ophthalmol. 2004; 88: 110-113.

15. Nita M, Strzaika-Mrozik B, Grzybowski A, Romaniuk W, Mazurek U. Ophthalmic transplantology: posterior segment of the eye - part II. Med Sci Monit. 2012; 18 (6): RA97-103.

16. Schmidt-Erfurth U, Hasan T, Gragoudas E, Michaud N, Flotte TJ, Birngruber R. Vascular targeting in photodynamic occlusion of subretinal vessels. Ophthalmology. 1994; 101 (12): 1953-1961.

17. Yannuzzi LA, Friedman R, Fine SL, Gass JD, Gitter KA, Orth DH, Singerman LJ. Symposium on age-related macular degeneration. Bull N Y Acad Med. 1988; 64 (9): 955-1013.