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Ilya Andreevich Popov, Sergey Anatol'evich Borzenok, Irina Nikolaevna Saburina, Patimat Magomedovna Arbukhanova, Dmitriy Ostrovskiy; Reconstruction of the Retinal Pigment Epithelium Cell Layer with Donor Cell Spheroid Microaggregates: Preclinical Study. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):177.
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© ARVO (1962-2015); The Authors (2016-present)
None of existing forms of retinal pigment epithelium (RPE) transplants is perfect. Previously we had shown how a new type of RPE transplant in the form of multicellular microaggregate (spheroid) can be constructed and recently we have tested the hypothesis that donor cadaver RPE spheroids can form a layer of RPE in vitro and investigated its properties.
We used adult cadaver donor eye globes (n=8, used in accordance with Helsinki Declaration) for establishing cell cultures of RPE (n=8) and obtaining RPE spheroids (n=128, 16 per culture) by 3D cell culturing of RPE cell suspension in hanging drops, as previously described (Borzenok et al., Siberian Branch of Russian Academy of Medical Sciences, 3(34), 2014). Spheroids had estimated number of 1000 cells per spheroid and mean diameter of 246,60±2,0 µm. Transplantable spheroids (n=98) were placed into the wells of 24-well plates (1 spheroid per well) with culture medium and cultivated for 30 days in standard conditions. Digital photos of wells were made and processed (CellSens, Olympus) to evaluate the rate of cell spreading and qualitative properties of formed cell layers. For control we used cell suspensions of the initial 2D cultures placed in the wells of 24 well plates (10 000 cells per well, 96 wells). Standard culture medium including DMEM/F12 with 10% of FBS and 1% of antibiotics was used at all steps.
Attached spheroids (91%) gave the spreading layer of cells around them. The area of cell layer increased in the first 10 days and reached a plateau soon after. The layer of cells gradually changed from multilayer in the center of former spheroid to monolayer at the mean distance of 164±32 µm of the center of spheroid and further, though not reaching the edge of well. The cells in the formed monolayer resembled native hexagonal RPE. Free-floating spheroids (9%) in day 4 acquired rough surface and changed no more. In the control group with fluent homogenous cell monolayer (90% of wells) cells had spindle-like stretched shape (p=0,005).
Spheroid of adult cadaver donor RPE can form limited layer of cells around the site of their attachment. The layer is inconsistent and has concentric areas of thickness gradually decreasing from center to periphery. The monolayer of cells formed by RPE spheroid implantation resembles native RPE layer more than it is in conventional cell suspension seeding.
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