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Purpose. To study experimentally in vitro secretion of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF) using intact and induced multi-potent mesenchymal limbal stem cells (MSCs) in three-dimensional culture (3D).Material and methods. MSCs were obtained by culturing of limbal fragments, isolated from the cadaveric human donor eyes, according to the medical technology of the S. Fyodorov Eye Microsurgery Federal State Institution. The phenotype of obtained cell culture was studied by the flow cytometry method.Stimulation of secretion of neurotrophic factors was performed via a two-step technique using non-specific activation factors: EGF, hbFGF, N2 additive, dibutyryl cAMP, NRG1-beta 1, PDGF, 3-isobutyl-1- methylxanthine. The 3D-cell spheroids were generated using agarose plates (3D Petri dishes, Microtissue, USA) for three comparative groups where: Group I – control, Group II – with the induction of spheroids at 1 day of cultivation; Group III – with induction of spheroids at 7 days of cultivation. The concentration of NGF and BDNF in the culture medium was studied using the enzyme linked immunosorbent assay (ELISA).Results. The induction of the 3D spheroids of limbal MSCs, that carried out on the 1st and 7th days of incubation, contributes to a significant increase in the production of NGF and BDNF, but subsequently a pronounced reduction in the secretion of these factors is observed. The conducting of an induction leads to a change in the morphology of spheroids: loss of compactness, the emergence of «fringed» (debris). Such changes indicate a non-viability of the obtained 3D-cell spheroids.Conclusion. The cellular spheroids, created from 2D-culture of intact limbal MSCs by the three-dimensional culture method, are capable in sufficient therapeutic concentrations spontaneously to synthesize NGF and BDNF, have the most optimal design for transplantation in extrabulbar and intraocular tissue niches of the eyeball, are a potential source of prolonged secretion of neural basis function in cell treatment of optic neuropathy.

At present it is accepted that glaucoma is a multi-factorial neurodegenerative disease, in which process there occur a death of retinal ganglion cells (RGC), progressive optic neuropathy and a visual field loss.In recent years, a number of large multicenter studies have convincingly shown that the effective reduction of intraocular pressure by medication and surgical methods does not guarantee a long-term stabilization of the glaucomatous process, and therefore a number of patients have a progression of the neurodegenerative process.This fact determines the necessity to search new ways of glaucoma optic neuropathy therapy, one of these can be the neuroprotection based on the methods of cell therapy.It is shown that the apoptosis is the primary mechanism of RGS death in glaucoma as in other neurodegenerative diseases. Currently found a large number of triggers of RGS apoptosis. One of the most important is the factor blocking axoplasmatic transport of neurotrophins.Neurotrophins are a family of structurally and functionally similar polypeptides that plays an important role in the differentiation, survival and regeneration of neurons.Numerous studies have shown that the neurotrophic factors in general, especially brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) significantly improves the RGC survival in experimental models of glaucoma.It is revealed that multipotent mesenchymal stromal cells (MMSC) can release a large number of bioactive factors, including neurotrophins, both in vivo, as well as in experiments in vitro.MMSC of eye limbus phenotypic match the bone marrowderived mesenchymal stromal cells. During MMSC culturing they secrete a wide variety of cytokines, interleukins, growth-factors. Therefore we consider the transplantation of allogenic limbus fragments as a candidate for cell therapy in the treatment of glaucomatous optic neuropathy.