Background and Aim: Application of tissue engineering is concerned tobe promising in all medical fields, including ophthalmology. The injuryof anterior and posterior eye sections affects multiple tissues: cornea, photoreceptors and retinal pigment epithelium (RPE) as well as a limbalzone that is the main source of eye stem cells for regeneration. Data aboutcell cultures from different eye regions are sometimes contradictory. Theaim of our work was to study 2D and 3D cell cultures of RPE and limbalzone.Methods: Primary cultures of RPE cells and multipotent mesenchymalstromal cells from the limbal zone (MMSC-L) were obtained from thepostmortal human eye. The study was conducted in accordance withofficially approved procedures and a special authorization existing underthe laws of Russian Federation. Cells were cultured for 4 passages understandard conditions (37°C, 5% CO2) in the growth medium consistingof DMEM/F12 (1:1), 2 mM/l L-glutamine, 50 μg/mL gentamicin. For RPEgrowth medium was supplemented with 20 ng/mL FGF, 20 ng/mL EGF, 1%solution of N2 and B27, and 5% FBS. For MMSC-L growth medium wassupplemented with 20 ng/mL bFGF, insulin-transferrin-selenite (1:100)and 10% FBS.
Spheroids were formed on agarose plates with micro-wellsmade using 3D Petri Dishes (Microtissue, USA). Time-lapse microscopywas performed with live imaging system Cell-IQ (CM Technologies,Finland). Immunophenotyping of cell cultures was conducted for thefollowing surface markers: CD34, CD45, CD90, CD105, CD14, CD11b,CD19, CD29. For immunocytochemical analysis cells in monolayer and7-day spheroids were fixed in 4% paraformaldehyde and were stainedwith antibodies to vimentin, ZO-1, N- and E-cadherin according torecommended protocols.Results: In 2D monolayer cultures, cells were highly adhesive to Petridish surface. RPE cell culture had polygonal cobblestone morphologywith tight intercellular junctional complexes that expressed ZO-1 proteincharacteristic of epithelial cells. By the 4th passage the phenotype of RPEcells changed: they acquired mesenchymal fibroblast-like morphologyand immunophenotype (CD105+, CD90+, CD45-, CD34-, CD11b-,CD19-), the level of vimentin expression was upregulated, synthesis ofmelanin was downregulated. MMSC-L during all the period in monolayerculture-maintained fibroblast-like morphology, expressed mesenchymalmarkers vimentin, N-cadherin, CD105, CD90, and CD29 almost didnot express epithelial marker E-cadherin. When placed in non-adhesiveconditions both cell types rapidly formed 3D spheroids. One of thecharacteristics of spheroids from cells of either epithelial or mesenchymalphenotype was the formation of several outer epithelial-like layers withadhesive E-cadherin+ (in case of RPE and MMSC-L) and tight ZO-1+ (incase of RPE) intercellular junctions. Moreover, spheroids from RPE andMMSC-L fused with each other and formed microtissues with epithelialcells on the surface and mesenchymal insight. 3D cell culture promoted aspontaneous mesenchymal-epithelial transition in MMSC-L, and revertedepithelial phenotype in RPE with upregulation of melanin synthesis.Conclusion: Our study revealed a gradual transition to mesenchymalphenotype in 2D culture and spontaneous restoration of epithelialphenotype in cell spheroids. Combination of epithelial and mesenchymalcharacteristics in studied cultures of RPE and MMSC-L indicates theirepithelial-mesenchymal plasticity, which makes these cells a promisingsource for regenerative medicine in the treatment of eye disorders.