Vascularization of bioengineered bone tissue constructs remains a challenging problem of regenerative medicine. Spheroids generated in 3D culture of adipose-derived stromal cells supplemented with inducing factors demonstrate stable characteristics and express of mesenchymal, endothelial, and osteoblasts markers, and represent a prototype of vascularized microtissue. Adipose-derived stromal cells spheroids induced to both angio- and osteogenic differentiation can be used in development of new innovative technologies for in vitro fabrication of vascularized bioequivalents for repair of large bone defects.

Предложена модель in vitro изучения биологической совместимости и токсичности полимерных материалов на культурах клеток стромы роговицы человека - кератоцитах (КЦ). Цель исследования - изучение возможности использования выделенных культур кератоцитов донора-трупа человека для оценки биосовместимости полимерных материалов. Методика. Из роговицы трупного донорского глаза получали первичную культуру КЦ и пересевали до 4-го пассажа. Фенотип КЦ подтверждали методом иммуноцитохимического окрашивания с выявлением основных клеточных маркеров. КЦ культивировали в присутствии образцов полимерных материалов - четырех модификаций бисфенол-А-глицедилметакрилата (бис-ГМА) по 24 образца каждого (4 экспериментальные группы). В качестве группы сравнения 1 использовали образцы из полиметилметакрилата (ПММА) идентичной геометрии (24 образца), группа сравнения 2 - КЦ, культивированные по стандартной методике без полимерных образцов (24 образца). КЦ в каждой группе распределяли по 24 лункам культурального планшета, культивировали на протяжении 6 сут., ежедневно КЦ извлекали из четырех лунок в каждой группе и подсчитывали. Анализируя динамику клеточного роста и качественное состояние образцов полимеров, делали вывод о типе биологической совместимости исследуемых материалов. Результаты. Все кривые клеточного роста имели восходящую S-образную форму, количество клеток статистически значимо увеличивалось со 2-х по 4-е сут. и замедлялось к 6-м сут. Среди исследованных материалов бис-ГМА № 3 проявил наименьшую способность обеспечивать адгезию культивируемых клеток, бис-ГМА № 1 и № 2 - наибольшую. Результаты статистически значимы. Заключение. Проведенные исследования показали высокую информативность использования предложенной методики для определения биологической совместимости искусственных материалов. Исходя из полученных результатов, все исследуемые материалы были отнесены к числу биологически активных. Образцы исследуемых материалов в клеточной культуре статистически значимо влияют на уровень клеточной адгезии и степень пролиферации. An in vitro model was proposed for studying biocompatibility and toxicity of polymeric materials in cultures of human corneal stromal cells, keratocytes (KCs). The aim of the present research was to study a possibility of using cultures of isolated human KCs to assess biocompatibility of polymeric materials. Materials and methods. The primary KC culture was obtained from donor’s eye cornea and cultured to the 4th passage. The KC phenotype was confirmed by immunocytochemical staining, and the major cell markers were identified. KCs were cultured in the presence of four modifications of bisphenol-A-glycidyl methacrylate (bis-HMA) polymeric materials (24 replicate samples for each modification). Polymethylmethacrylate (PMMA) samples of identical geometry were used in the first comparison group (24 samples). In the second comparison group, KCs were cultured according to a standard procedure without polymer samples (24 wells). In each group, KCs were distributed to 24 wells of the culture plate and cultured for 6 days; cells were counted daily. Based on the dynamics of cell growth and qualitative condition of polymer samples, we made a conclusion about the type of biological compatibility of the materials under study. Results. All cell growth curves had an upward S shape; the number of cells increased statistically significantly from day 2 to day 4 (p <0.05) and slowed by day 6 (p>0.05). Among the studied materials, bis-HMA #3 showed the weakest ability (p <0.05) and bis-HMA #1 and #2 - the greatest ability (p<0.05) to provide adhesion of cultured cells. Conclusion. The study showed a high informative value of the proposed method for determining biological compatibility of artificial materials. All studied materials were classified as biologically active. Samples of the studied materials statistically significantly affected cell adhesion and proliferation in the cell culture.

Функциональная активность меланоцитов обусловливает защитные свойства кожи против воздействия ультрафиолета, вызывающего фотостарение. Однако изучение меланоцитов in vivo затруднено тем, что необходимо использовать животные модели и трудоемкие методы анализа, а культивирование клеток в монослойной культуре in vitro сопряжено с потерей тканеспецифичных маркеров клеток. Данная работа посвящена получению и изучению сфероидов из меланоцитов, так как 3D культивирование меланоцитов в виде сфероидов может сохранить их фенотип и функциональность. Исследование проводили на первичной культуре меланоцитов кожи человека. Клетки культивировали в монослое в полной ростовой среде до 4 пассажа. Далее клетки помещали на агарозные планшеты с микролунками в посевной плотности 3,3 х 10 кл./мл. Анализ полученных сфероидов производили с помощью фотометрии, иммуноцитохимии и ПЦР в реальном времени. Было показано, что, при культивировании в монослое, к 4 пассажу снижалось количество синтезируемого меланина. Тогда как в 3D условиях меланоциты формировали компактные сфероиды, внутри которых в процессе культивирования не только сохранялся синтез, но и накапливался меланин. Была выявлена экспрессия специфических генов TYR и MCR1, и увеличивался синтез белков, участвующих в меланогенезе - gp100 и MITF. Таким образом, в данном исследовании было показано, что меланоциты in vitro способны формировать длительно живущие, жизнеспособные 3D структуры - сфероиды, с сохранением фенотипа и синтеза тканеспецифичных маркеров. Поэтому данные сфероиды могут быть успешно использованы как тест-системы для оценки эффективности препаратов, направленных на регуляцию уровня пигментации кожи. The functional activity of melanocytes determines protective properties of the skin against the effect of ultraviolet radiation, which causes photo-aging. However, studying melanocytes in vivo and in an in vitro monolayer culture is difficult because animal models and laborious analytical methods are required, and the culturing is associated with loss of tissue-specific cell markers. Since 3D cultivation of melanocytes in the form of spheroids can preserve their phenotype and functionality, this work focused on obtaining and studying melanocyte spheroids. The study was conducted using a primary culture of human skin melanocytes. Cells were cultured in a monolayer to the fourth passage. Then these cells were placed in agarose plates with microwells at the cell suspension concentration of 3.3 х 10 in vitro . These features make the melanocyte spheroids a convenient test-system for studying toxicity and efficiency of drugs targeted at regulation of skin pigmentation.

One of the essential goals in regenerative medicine is microvascularization which enables an effective blood supply within de novo constructed tissues and organs. In our study, we used two common multipotent mesenchymal stromal cell (MMSC) sources (subcutaneous adipose tissue and Wharton's jelly of the umbilical cord) where is a subpopulation of endothelial precursors. In the medium supplemented with VEGF, the 3D cultures of UC MMSCs and ADSCs promoted the endothelial cell differentiation. To evaluate their ability to form a capillary-like network, we encapsulated spheroids within non-modified and PEGylated fibrin hydrogels. The PEGylated hydrogel supported better the formation of multibranched cords than the pure fibrin gel. Analysis of tubule growth rate, length, and branching showed that the differentiated ADSCs had higher angiogenic potential than the differentiated hUC MMSCs. Our study can be a basis for the development of new strategies in tissue engineering and treatment of vascular diseases.

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.

На сегодняшний день показано, что стволовые и прогениторные клетки открывают новые возможности для восстановления поврежденных тканей глаза. Основным источником стволовых клеток для регенерации является сформированная в большей степени производными нервного гребня лимбальная зона глаза. Данное исследование посвящено разработке инновационной технологии создания 3D сфероидов из мультипотентных мезенхимных стромальных клеток лимба (ММСК-Л). Показано, что в 3D условиях культивирования ММСК-Л за счет компактизации и мезенхимо-эпителиального перехода самоорганизуются в клеточные репаративные модули. Сформированные сфероиды ММСК-Л сохраняют и поддерживают недифференцированную популяцию стволовых и прогениторных клеток лимба, что подтверждает экспрессия таких факторов плюрипотентности, как Oct4, Sox2 и Nanog. Синтезируемый клетками сфероидов внеклеточный матрикс позволяет поддерживать функциональный потенциал ММСК-Л, участвующих в регенерации как переднего, так, вероятно, и заднего отрезков глаза.

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

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.

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.

Experiment was conducted on 40 rats of Wister line. On the artificially reproduced experimental model autogenic mesenchimal stem cells (MSC) of adipose tissue were inoculated in space between bone autograph of tibia and mandible. MSC wasn't inoculated in the comparison group. Formation of a new bone substance in space between an autograph and mandible bone was observed. It was clear that after 120 days (180 days), there was a statistically significant decline of the area occupied by an immature fibroreticular bone. Described phenomenon, presumably, could be explained as a result of decline of the number of active cells in the population of inoculated MSC according to phenomenon of limited number divisions of cells on telomeres, described by Hayflick L. and Moorhead P.S.

We developed an original reproducible 3D-technology for preparation of single dormant microspheres consisting of 2000 somatic cells. The dynamics of microsphere assembly from mesenchymal and epithelial cells of retinal pigment epithelium was traced using time-lapse microscopy: formation of a loose aggregate over 24 h followed by its gradual consolidation and formation of a compact viable microsphere with a diameter of 100–150 μ by day 7. The cell number in the formed microspheres remains unchanged. Reactivation observed upon fusion of epithelial and/or mesenchymal microspheres results in the formation of a united compact microtissue. The fusion dynamics reproduces spherogenesis irrespective of the initial amount of co-cultured microspheres. Reactivation via two-step induced angiogenesis opens new prospects for production of vascularized microspheres and microtissues.

In experiments on rats CD comparative studies have been carried out which is the effect of osteoplastic material NORIAN CRS, the rate of bone regeneration after mandibular dental implants (titanium screw) and the combined effect of these processes NORIAN CRS protein and protein (Emdogein). Functionality and maturity of the newly formed bone tissue was evaluated by histological and biochemical methods. The results suggest that the use of osteoplastic material NORIAN CRS is the 120 days leading to almost complete regeneration of bone tissue. It was also found that the combined use of NORIAN CRS and protein Emdogein impractical because adding Emdogein not accelerate bone regenerative process, but in the early postoperative period may cause an inflammatory reaction.

The article presents the developed methods for the preparation of multicellular functional modules (spheroids) from dissociated cells of various tissues and organs of humans and mammals. Spheroids were obtained and studied from the stroma of adipose tissue of mouse and human, bone marrow and the main substance of umbilical cord of human. Contents of significant reserves vasculogenic stroma and immature endothelial progenitor cells is a good model for studying angio-and vasculogenesis. In the model of lower limb ischemia has been shown high viability of cells comprising the spheroids, and their active involvement in vasculo-and angiogenesis in comparison with the cell suspension. The authors have shown the possibility of induced vasculogenesis in the spheroid formed from dissociated cells of the stromal of the main substance of human umbilical cord (SCUC).