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The disruption of blood flow through the retinal vessels is undoubtedly a stressful situation for the body. In response to any stress, the body produces heat shock proteins or so-called stress proteins. Moreover, a number of modern studies indicate that HSP may act as autoantigens, which are components of the immune system involved in the pathogenesis of vascular pathologies. By now, a number of certain heat shock proteins (HSP 60, 70) have already been proven to play a significant role in the pathogenesis of atherosclerosis as well as various circulatory disorders in the cerebral vessels. In addition, the possibility of using the data on the content and concentration of certain HSPs and antibodies to them in the blood plasma of patients as diagnostic and prognostic markers of systemic vascular diseases is considered. On the visual organ side, HSPs have been found to have damaging effects on retinal ganglion cells and, together with antibodies produced in response to their presence, are involved in the pathogenesis of glaucoma. However, there is currently no data on the role of HSP in the pathogenesis of retinal blood flow disorders and the possibility of using these molecules as diagnostic or prognostic markers of such conditions.

Visual impairment in elderly people is a serious problem that significantly affects the quality of life of millions people around the world. The magnitude of this problem is becoming increasingly apparent as the population ages and the number of older people increases. Age-related macular degeneration (AMD) is the third leading cause of blindness worldwide and the main cause of vision loss in people over 60 years. It is expected that AMD will affect about 288 million people by 2040. AMD is a multifactorial disease with a progressive course. The arised dystrophic changes in the retina cannot be reversed by any of the known treatment methods. A lot of research and effort has already been invested in identifying various biomarkers for predicting the incidence rate, identifying people at risk, finding out the pathogenetic mechanisms of this disease, and finding effective methods of treatment and prevention.Aging is the basis of pathological changes that occur during AMD. Aging biomarkers are measurable vital signs that qualitatively and quantitatively change with the age of the body. DNA methylation is a molecular mechanism that is a potential biomarker of aging. Sirtuins indirectly participate in this process, regulating the activity of the DNMT1 enzyme. The article discusses current knowledge of the mechanisms underlying the action of sirtuins (Sirtuins / SIRT), with an emphasis on SIRT1. Analysis of the pathophysiological action of sirtuins can affect the prevention and treatment of pathological eye changes associated with AMD. The article provides literature sources containing the results of studies of the effect of SIRT1 as a marker of aging in body tissues. SIRT1 is an attractive candidate for developing therapeutic strategies preventing early eye aging, in particular, age-associated diseases such as AMD The impact on the genetic mechanisms of this disease is a promising direction in treatment.

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.

Aim. To develop technology to create 3D-spheroid multipotent mesenchymal stem cells (MMSC) of limbal cadaveric human eyes, capable of safe and long-term secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF).Materials and methods. MMSC were obtained by cultivation of limbal fragments, released from cadaveric donor human eye. Cultivation was carried out in DMEM/F12 medium, supplemented with L-glutamine, penicillin, streptomycin, amphotericin B, HEPES, insulin, dexamethasone and 10 vol.% FBS under standard conditions (5% СО2, 37 °C), medium change was performed every 3 days. To determine the phenotype of the received cell culture the method of immunophenotyping by marker proteins to MMSC (CD73, CD105, CD19, CD90, CD133) was used. Stimulation of neurotrophic factor secretion was performed via a twostep procedure. 3D-cell spheroids were created with the help of agarous plates for three groups of comparison, where group I was control group, spheroids of intact 2D-culture MMSK; group II – spheroids of previously induced 2D-culture MMSC; group III – spheroids of 2D-MMSC induced on the 1st day of cultivation. Cell cultures supernatants were selected in different periods for NGF and BDNF follow-up study by ELISA procedure.Results. Induction of 3D-spheroids of limbal MMSC promotes short-term increase of the level of BDNF and NGF, but further, the secretion of these factors significantly decreases. Induction leads to a change in the morphology of spheroids: loss of compactness and emergence of «fringed» (debris). Such changes indicate of frailty of received constructions. Spheroids from previously induced MMSC are capable of stable NTF secretion, but the level of secretion is much less as compared to the control group.Conclusion. 3D-cell culture of intact 2Dculture of limbal MMSC can be considered as cellular medication for a safe and long-term neuroprotection in optic neuropathy treatment.

Modern techniques of laser microsurgery of cell spheroids were used to develop a new simple reproducible model for studying repair and regeneration in vitro. Nanosecond laser pulses (wavelength 355 nm, frequency 100 Hz, pulse duration 2 ns) were applied to perform a microdissection of the outer and the inner zones of human bone marrow multipotent mesenchymal stromal cells (BM MMSC) spheroids. To achieve effective dissection and preservation of spheroid viability, the energy of laser pulses was optimized and adjusted in the range 7-9 μJ. After microdissection, the edges of the wound surface opened and the angular opening reached a value of more than 180°. The destruction of the initial spheroid structure was observed in the wound area, with surviving cells changing their shape into a round one. Partial restoration of a spheroid form took place in the first six hours. The complete structure restoration accompanying the reparative processes occurred gradually over seven days due to remodelling of surviving cells.

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.

Purpose. To study prerequisites for a development of artificial cornea bioengineered design based on recombinant spidroin tissue matrix by behavior evaluation of 2D (planar) and 3D cell (threedimensional) cultures on its surface.Material and methods. We studied epithelioid and stromal primary cell cultures (MSC-L) received from the limbal zone of post-mortem donor eyes. Cells were seeded on Petri dishes and on cavities of cultural trays (Corning, USA). To get spheroid structures the cells after the second passage underwent the centrifuge and were seeded on agarous trays then were cultivated in thermostatic chamber (Cell-IQ, Chip Man Technologies, Finland) under standard conditions (37° C, 5% CO2). Control over cell growth and morphology in trays was conducted under inverted microscope CKX41 (Olympus, Japan). To count the cell quantity and their viability the automatic cell counter Countess (Invitrogen, USA) was used, to analyze the surface proteins expression the flow cytofluorimetry was applied. For matrices colonization we used the 3rd passage MSC-L and 7-day spheroids of MSC-L origin. To evaluate 2D and 3D cell cultures growth on the surface of membranous matrices of recombinant spidroin, to estimate its non-toxicity and adhesiveness the immunohistochemistry, light time-lapse microscopy (Cell-IQ, Chip Man Technologies, Finland), laser scanning confocal microscopy (FluoView FV10i, Olympus, Japan) and raster electronic microscopy (CamScan, Japan) were incorporated.Results. Few hours after cell seeding there was active cells’ attachment to the substrate. Attached cells were characterized by rounded, oval or polygonal ordonnance. 24 hours later bipolar elongated cells and islets of migrating epithelioid cells appearance were observed. In the incubation process under gravity force the spheroids were accumulated predominantly in the central zone of the matrix, 2 hours later an active migration of spheroids surface zone epithelioid cells was registered on the membrane. After 24 hours of incubation all seeded on the surface of membranous matrix cells possessed a mesenchyme-like phenotype. Spheroids had an ability to merge limitlessly, later we observed a new microtissue formation with epithelioid cells on the surface and mesenchyme-like cells in the central area. Both solitary spheroids and merger-derived microtissue contained epithelial and mesenchymal components as well as regularly organized fibrils of extracellular matrix.Conclusions. According to aforementioned data the development of artificial cornea bioengineered cell-tissue constructions based on the technology of 3D cell spheroids cultivation derived from multipotent stem cells of the limbus and spidroin matrix presents a promising prospect requiring a further profound investigation.

Aim. To study in experiment the criteria for transplantability of multicellular spheroid microaggregates of retinal pigment epithelium (RPE), prepared by the method of 3D cell culture. Materials and Methods. 11 donor eyes (6 of adrenaline index «A», 5 of index «B») were used as a source of RPE cell cultures (group «A» – 6 cultures, group «B» – 5 cultures), of which over 2000 RPE spheroids were obtained by the method of three-dimensional cell culture. 1760 spheroids of them were selected for transplantability investigation (960 – group «A», 800 – group «B»). Among the selected spheroids were equal numbers of spheroids of different morphology («smooth» and «rough») and of the initial cell seeding number (500, 1000, 5000, 25 000, 125 000 cells per hanging drop). We were taking out 12 spheroids of group «A» and 10 spheroids of group «B» of the 3D culture in terms of 7, 14, 21, 28 days of 3D culture to assess their viability. We were transferring the same number of spheroids in the same terms from 3D to 2D culture conditions to assess their adhesive properties. Viability of cells within spheroids was determined using the Trypan blue exclusion. The presence or absence of adhesion was determined by microscopic observation.Results. «Smooth» spheroids of 7 and 14 days of pretransplantation cultivation and derived from hanging drops containing 500 and 1000 cells showed the highest transplantability (cell viability varied from 0.83 ± 0.38 to 0.94 ± 0.24, a 100% adhesion). «Rough» spheroids were untransplantable in all variants, despite their partial preservation of viability (in comparison to “smooth” ones p < 0.05). 21 and 28 days of pretransplantation culturing and high cell seeding numbers signifi cantly lowered transplantability of obtained spheroids (p > 0.05 for low cell numbers, p < 0.05 for the high ones). Differences in adrenaline indexes A and B of donor eyes which were the primary sources of cellular material had no effect (p > 0.05) on resulting spheroids transplantability.Conclusion. Among RPE spheroids obtained by our method the spheroids cultivated in a 3D environment for 7 to 14 days prior to transplantation and derived from hanging drops containing 500 and 1000 RPE cells showed the highest transplantability.

The article reviewed biopolymer materials to construct matrices of the bioengineered tissue structures used at present. Polymer materials such as alginate, chitozan, collagen, spidroin were analyzed. There were shown advantages and disadvantages of these materials to construct matrices of the bioengineering construction of the artificial cornea. Spidroin is a recombinant analog of dragline spider silk protein, and in our opinion it is the most promising and adequate to construct matrices of the bioengineering construction of the artificial cornea. Spidroin is the material of choice to construct 3D matrices of the bioengineering construction of the artificial cornea due to its properties such as high adhesive properties, pronounced mechanical properties, a biological inertness, a biocompatibility, a resistance to environmental conditions and an absolute transparency.

Aim. To compare the effectiveness of MSC with different degree of ontogenetic maturity (MSC bone marrow – MSC BM and MSC umbilical cord – MSC UC) on regenerative processes in injured liver. Methods. In 4 groups of experiments on Wistar rats (n = 80) with a model of fibrotic toxic liver damage (FLD) it was studied the effect of MSCs with different degree of ontogenetic maturity on recovery processes at the regeneration of damaged liver: 1 gr. – Control, 2 gr. and 3 gr. introduction of MSC BM, included in Sphero®GEL-long in doses of 2.5 ×106 and 5.0 x 106 cells, respectively, and 4 gr. – introduction of MSC UC in the form of cell-spheroids (8–10 × 105 cells). The cells were injected into the damaged liver in 7 days after the end of FDL-modeling. The effect of cell therapy was studied during 180 days. The effectiveness of corrective therapy was evaluated by the results of functional and morphological investigations of livers (histological control of parenchymal and nonparenchy- mal liver tissue). Results. MSC BM in both doses and MSC UC contributed to a more rapid normalization of liver enzyme indices compared with the control (1 gr.), but the differences in the rate of recovery of disturbed enzymatic liver functions between groups 2, 3 and 4 – were absent. In 90 days after the cell application it was determined a more pronounced recovery activity of cells in groups 3 and 4; in 180 days the more pronounced activation of recovery processes was observed in group 3; but in group 4 the sclerotic processes were more pro- nounced in this period. Conclusion. For the induction of recovery processes in damage liver it is advisable not to use the MSC UC, but to use MSC BM in the Sphero®GEL, because MSC BM exert not only local but also systemic immune-regulatory effect, increasing the pool of T-reg. cells, which are additional carriers of regenera- tion information in organism.

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3–4 days of human BM stromal cell cultivation in the presence of 2–20 μg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3–4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus–bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation.

We studied expression of dystrophin in skeletal muscles of C57BL/10J-mdx mice after transplantation of human embryonic and fetal myoblasts and bone marrow stromal cells. Dystrophin-positive areas corresponding to the location of transplanted cell were detected in muscles of all recipient mice after transplantation of different cell cultures, but the distribution of dystrophin characteristic of normal muscle fibers was detected only after transplantation of embryonic myoblasts. Dystrophin distribution in muscle fibers after transplantation of fetal myoblasts and bone marrow stromal cells was atypical. [ABSTRACT FROM AUTHOR]