CELL CULTURES Information Bulletin. Issue 32, St. Petersburg, 2016

CONTENTS N.P. Teryukova, E.I..Sakhenberg, V.A. Ivanov, S.A. Snopov. The comparison of morphological and functional properties of the monolayer hepatoma zajdela clonal lines with features of stem cells and progenitor cells. pp. 3–14. T.D.Smirnova, S.S. Smirnova, K.V. Sivak, E.M. Eropkina, M.Y. Eropkin. Human cell line ECV-304 as a model for the study of the effect of antioxidant preparations on the cellular proliferation and apoptosis under infection with influenza a virus and in the presence of bacterial lipo-polysaccharide. pp. 14–28. V.I.Turilova, T.K.Yakovleva Functional activity of nucleolus organizer regions in metaphase chromosomes of human multiple myeloma cell lines. pp. 28–43. I.A. Suetina, M.C. Mezentseva, E.A. Gushchina, F.A. Lisitsyn, L.I. Rusu, O.A. Lopatina, E.L. Firsova, J.F.Tyson, M.E. Johnson, Xing B., S.A. Ostroumov. Metal nanoparticles influence on the viability and morphofunctional characteristics of cultured human and animal cells. pp. 43–53. V.P. Pashchenko. Experience using tissue and cell cultures in clinical practice. pp. 53–62. V.M. Semenova, D.М. Yehorova, L.P. Stayno. Possibilities of a tissue culture method in a research of biological properties of stem tumor cells of a malignant brain gliomas pp. 62–81. N.B. Bildyug. On the problem of cardiomyocyte differentiation methods. pp. 82–95. S.A. Alexandrova, Y.A. Naschekina N.V., Tsupkina. Methodological approaches of creating tissue engineering grafts to restore defects of bone and cartilage tissues (the experience of the Institute of Cytology RAS). pp. 95–104. D.E. Bobkov. Lysophosphatidic acid as regulator of normal and pathological processes. pp. 104–109. THE COMPARISON OF MORPHOLOGICAL AND FUNCTIONAL PROPERTIES OF THE MONOLAYER HEPATOMA ZAJDELA CLONAL LINES WITH FEATURES OF STEM CELLS AND PROGENITOR CELLS N.P. Teryukova*, E.I..Sakhenberg, V.A. Ivanov, S.A. Snopov FGBUN Institute of Cytology RAS, St. Petersburg * npter@yandex.ru We continued our study of the hepatoma Zaydela clonal lines established by us after explantation of the ascitic tumor cells into cell culture. Previously, we have shown that the cells of line 5F and line 1E are respectively originated from holoclons and meroclons and reveal the features of tumor stem cells (TSC) and tumor progenitor cells (TPC). According to our morphometric analysis, the two cell lines differed significantly in cell morphology, in a whole cell size, in a size of the nuclei and in nuclear-cytoplasmic ratio. Using the wound-healing (scratch) assay and video microscopy we have revealed for the first time fundamental differences in migration abilities of the cells. Thus, TSC 5F line cells showed autonomous behavior and used the amoeboid movement type for an active migration to the scratch; whereas TPC 1E line formed tight junctions between cells and produced a rapid scratch closure via collective cell migration. The results suggest that migrating cells of the line 5F represent the population of metastatic TSC. In addition, we are the first who have identified a nuclear localization of the intracellular domain of epithelial cell adhesion molecule (EpCAM) in the cells of cultured lines; we believe that localization indicates the activation of EpCAM-signaling pathway in TSC and TPC of a metastatic tumor. Key words:  hepatoma, metastasis, tumor stem cells, epithelial cell adhesion molecule, cell migration, amoeboid motion type. HUMAN CELL LINE ECV-304 AS A MODEL FOR THE STUDY OF THE EFFECT OF ANTIOXIDANT PREPARATIONS ON THE CELLULAR PROLIFERATION AND APOPTOSIS UNDER INFECTION WITH INFLUENZA A VIRUS AND IN THE PRESENCE OF BACTERIAL LIPO-POLYSACCHARIDE T.D.Smirnova1, S.S. Smirnova1,2, K.V. Sivak1, E.M. Eropkina1, M.Y. Eropkin1 1 1Research Institute of Influenza of the Minisrty of Health care of Russia, 2 Peter the Great St.-Petersburg Polytechnic University, St.-Petersburg cellcultures@influenza.spb.ru Influenza illness provoked by influenza virus A has sometimes a severe form that could be aggravated with secondary bacterial infection. During the latter, bacterial endotoxins may be released into the blood stream, especially the lipo-polysaccharide of the Gram-negative bacteria. The purpose of present work was to study the influence of some preparations having antioxidant, anti-inflamatory and angio-protective activities on the proliferation and apoptosis of cultured endotheliocytes of the line ECV-304 which were infected with different doses of influenza A virus in the alone and in the presence of bacterial lipo-polysaccharide. The following preparations were tested: reduced glutathione, trolox, carvedilol, resveratrol, dicumarin, nitrendipine, rutin and alpizarin. Cell proliferation was inhibited after inoculation of high infectious doses of a virus and it has grown to the normal figures after the addition of glutathione, resveratrol, alpizarin and rutin. Dicumarin and N-acetyl-cysteine enhanced cell proliferation to the lower extent. At the same time, the enhanced level of cellular proliferation which was observed during the cellular infection with very low infectious doses has diminished to the control level under effect of all studied substances except nitrendipine. Similar results were obtained when cells were infected in the presence of bacterial lipo-polysaccharide. The index of apoptosis in cells under study has grown in the most experimental designs. Only three preparations - rutin, alpizarin and glutathione diminished apoptosis level in infected cells and also in the presence of lipo-polysaccharide. The obtained results evidence that nearly all tested pharmaceutical compounds can normalize the physiological state of cells under influenza virus infection by means of reduction of the excessive cellular proliferation and the elimination of cells by the apoptosis. Key words:  endothelium cells ECV-304, influenza virus A, LPS, antioxidants, proliferation, apoptosis FUNCTIONAL ACTIVITY OF NUCLEOLUS ORGANIZER REGIONS IN METAPHASE CHROMOSOMES OF HUMAN MULTIPLE MYELOMA CELL LINES V.I.Turilova, T.K.Yakovleva FGBUN Institute of Cytology RAS, St.Petersburg urilova@mail.cytspb.rssi.ru The number of ribosomal RNA (rRNA) gene repeats and their activity determine structural and functional nucleolus organization (as well as nuclear architecture) and ensure cell viability and environmental response. Dysregulation of rRNA synthesis and ribosomal biogenesis disturbs cell homeostasis and plays a crucial role in the development of malignancy. Human tumor cell lines are a convenient model for studying various aspects of neoplastic transformation including functional status of nucleolus organizer regions (NOR) of chromosomes. NOR activity in metaphase chromosomes of human multiple myeloma (MM) cell lines has been studied by standard silver staining. MM cell lines that differ in the degree of differentiation and cell ploidy level varied as well in the number, as in the composition, the total size and associative ability of Ag-positive NOR (AgNOR) chromosomes. It has been shown that the number of AgNOR-bearing chromosomes ranged from 4 (U266) to 8 (L363) in near-diploid cell lines, while in near-triploid cell lines it was 12 (RPMI 8226) and 14 (U1996). The lowest average total size of AgNORs - 7.19 ± 0.03 arbitrary units - was detected in U266 cells, the highest - 24.82 ± 0.09 arbitrary units - in U1996 cells. The proportion of cells with associations of AgNOR-bearing chromosomes ranged from 0 (Karpas 707) to 41% (U1996), and the proportion of AgNOR-bearing chromosomes involved in associations varied from 1.5% (U266) to 11.25% (L363). Comparison of the AgNOR average total size between MM cells that continuously maintaining in vitro, and the PHA-stimulated peripheral blood lymphocytes of healthy individuals (literature data) shows that MM cell lines have low copy number of active ribosomal genes, which may correspond to the differentiation level of MM cells in vivo. Key words:  human multiple myeloma cell lines: L363, Karpas 707, RPMI 8226, U266, U1996, Ag-positive nucleolus organizer regions of metaphase chromosomes, AgNOR size, cluster size polymorphism of active ribosomal genes, satellite associations of human acrocentric chromosomes METAL NANOPARTICLES INFLUENCE ON THE VIABILITY AND MORPHOFUNCTIONAL CHARACTERISTICS OF CULTURED HUMAN AND ANIMAL CELLS I.A. Suetina1, M.C. Mezentseva1, E.A. Gushchina1, F.A. Lisitsyn1, L.I. Rusu1, O.A. Lopatina1, E.L. Firsova1, J.F.Tyson2, M.E. Johnson2, Xing B.2, S.A. Ostroumov3 1 Federal Reseach Center N.F.Gamaley of Epidemiology and Microbiology D. I. Ivanovsky Institute of Virology. Ministry of health of the Russian Federation, Moscow 2 University of Massachusetts, Amherst, MA, U.S.A. 3 Moscow State University. Lengory, 119991, Moscow, Russia cells@rambler.ru To study the toxic effects of nanoparticles on the morphofunctional characteristics of the cells, a comparative testing of aqueous suspensions of nanoparticles of CuO and Fe2O3, on the primary trypsinized cell cultures of fibroblasts of embryonic chickens and lambs testicles, transpartable cultures of human cells (HEF-T and A549) and animals (BHK-21, Vero). Displaying different toxic effects on the cells of the test drugs. Using the MTT assay showed a reduction in cell viability during the incubation with the studied agents. The highest cytotoxicity after 48 hours of incubation was found with copper oxide nanoparticles, the lowest - from nanoparticles of iron oxide. CuO nanoparticles in aqueous suspension caused significant cell death at concentrations up to 1 µg\ml, whereas the sample Fe2O3 - in concentration 500-125 µg/ml, that may indicate the degree of exposure depending on their physicochemical properties. Results of transmission electron microscopy showed pathological changes in cell morphology during interaction for 48 hours with the studied Fe2O3 nanoparticles at 100 µg\l concentration. Explored the potential impact of sub-toxic concentrations of these nanoparticles on the parameters immunity. It is noted that the CuO nanoparticles inhibited gene expression and IFN-λ3 , and promote transcriptional activation of IL-18, IL-2, IL-6, TNF-α. Fe2O3 nanoparticles activated IFN-α , IL-2 and IL-1β, IL-6, IL-10 and TNF-α in the same cells. Key words:  nanoparticles of metal oxide, cultured human and animal cells EXPERIENCE USING TISSUE AND CELL CULTURES IN CLINICAL PRACTICE V.P. Pashchenko Northern State Medical University, Arkhangelsk paschenkow@mail.ru The article presents the results of a study of morphological and functional characteristics of the tissue culture of the bronchial mucosa fragments obtained by bronchoscopy in patientswith chronic diseases of bronchial tubes and lungs, as well as fragments of kidney tissue cultures of urological patients. Estimated size of the area of tissue growth, raised the number of cells, mitotic activity, abnormal mitosis and the number of abnormal cells. The data allowed to specify the diagnosis and forecasts of the outcome of the chronic diseases of the lungs and bronchi and evaluate the reparative potential of kidney tissue cells that mattered in the choice of tactics of treatment of patients.Using cell culture allowed to establish the effect of activating serum obtained from patients with alcoholism in the 1st month of abstinence, the growth of human embryonic fibroblast cells that should be considered in the pathogenesis of this disease and the pathogenesis of alcohol embryopathy. Key words:  tissue and cell cultures, lung, bronchus, kidney, diagnostics, alcoholism, alcoholic embryopathy POSSIBILITIES OF A TISSUE CULTURE METHOD IN A RESEARCH OF BIOLOGICAL PROPERTIES OF STEM TUMOR CELLS OF A MALIGNANT BRAIN GLIOMAS V.M. Semenova, D.М. Yehorova, L.P. Stayno SI "Institute of neurosurgery n.acad. A. Romodanov NAMS of Ukraine" This review presents recent data on the biological properties of tumor stem cells in the malignant brain glioma, their role in a histogenesis of these tumors and relationship with neuranal stem cells of neurogenerating zones of a brain. The possibilities of use of a method of cultivation in studying of a neurosphere formation - fundamental property of stem tumor cells are shown. Methodical approaches to receiving neurospheres in the in vitro conditions, and also ways of identification of stem tumor cells as a part of neurospheres are given. Results of an assessment of activity of a neurosphere formation in culture depending on degree of an anaplaziya of initial tumors taking into account clinical outcomes of a disease at neurooncological patients are presented. Prospects of practical use of the cultivated neurospheres for the purpose of search the antiblastic of preparations with selective impact on stem tumor cells of gliomas are described. Key words:  cancer stem cells, neuronal stem cells, neurosphere, brain tumor glioma, cell culture ON THE PROBLEM OF CARDIOMYOCYTE DIFFERENTIATION METHODS N.B. Bildyug FGBUN Institute of Cytology RAS, St. Petersburg relapse@yandex.ru There are a lot of methods of cardiomyocyte differentiation from different cells known up to date. However, approaches available and the methods of their effectiveness estimation are limited. The overview of main approaches and limitations associated with the in vitro and in vivo cardiomyocyte differentiation from different cell types is provided, including embryonic stem cells, cardiac stem cells, induced pluripotent stem cells and fibroblasts. Key words:  cardiomyocytes, differentiation methods, stem cells, fibroblasts METHODOLOGICAL APPROACHES OF CREATING TISSUE ENGINEERING GRAFTS TO RESTORE DEFECTS OF BONE AND CARTILAGE TISSUES (THE EXPERIENCE OF THE INSTITUTE OF CYTOLOGY RAS) S.A. Alexandrova1, Y.A. Naschekina1,2, N.V. Tsupkina1 1 Institute of Cytology RAS, St. Petersburg 2 Peter the Great St. Petersburg Polytechnic University, Dep. Medical Physics alekssvet2205@gmail.com The article describes an overview of the methodological approaches to the design of tissue-engineering grafts to restore defects of bone and cartilage tissues, developed by researches of the Institute of Cytology Russian Academy of Sciences. The conditions required for the adhesion and growth of rabbit multipotent mesenchymal stromal bone marrow cells on the different matrixes (type I collagen, demineralized bone matrix, poly(L,L)-lactid and biositall) were defined, methods of combining cells and three-dimensional matrixes were developed, the obtained tissue-engineering structures were tested by laboratory animals, that showed the possibility of their using in the clinic. Key words:  multipotent mesenchymal bone marrow stromal cells, osteogenic-, chondrogenic differentiation, type I collagen, demineralized bone matrix, biositall, poly(L,L)-lactid, tissue engineering grafts LYSOPHOSPHATIDIC ACID AS REGULATOR OF NORMAL AND PATHOLOGICAL PROCESSES D.E. Bobkov Institute of Cytology RAS, St. Petersburg bobkovde@yandex.ru This review presents the results of studies on the role of lysophosphatidic acid in the regulation of normal and pathological processes.Lysophosphatidicacid (LPA) is a phospholipid mediator signaling molecule. LPA plays an important role in inflammatory and wound healing processes due to its ability to regulate a variety of cellular functions, such as migration, proliferation and differentiation. LPA acts on the cell as autocrine or paracrine signaling molecule. LPA molecules interact on cell surface with specific G-protein bound receptors and activate a series of intracellular signaling cascades, including Rho pathway. LPA also participates in the pathogenesis of several diseases, such as atherosclerosis, fibrosis, respiratory disorders, and cancer. Key words:  lysophosphatidic acid, Rho signaling pathway, carcinogenesis