Influence of Fucoxanthin on Proliferative Activity of Human Melanocyte Culture.

We studied the effect of algae pigment fucoxanthin on proliferative activity of melanocyte culture from human skin. Fucoxanthin in high concentrations can be cytotoxic, which was confirmed by changes in melanocyte morphology and a decrease in their proliferative activity.

Induction of Vasculo- and Osteogenesis in Spheroids Formed by Adipose-Derived Stromal Cells.

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.

Characteristics of Trophoblasts in Long-Term Culture.

We analyzed more than 40 cytotrophoblast cultures derived from cell islets that grew from trypsinized tissue fragments of placental microvilli. Phenotypic variability of trophoblasts was demonstrated. Changes in trophoblast morphology from epithelium-like or oval cells to bipolar and spindle-shaped or twisted and then to mesenchymal-like cells as well as intensive expression of cytokeratin-7 and vimentin attested to epithelial-mesenchymal transition of trophoblasts during in vitro culturing. Analysis of the expression of specific markers in long-term trophoblast culture (≥7 passages) revealed the possibility of culture contamination with other non-trophoblast cells including fibroblasts. High risk of trophoblast culture contamination with rapidly growing cells necessitates regular control of the cultures used in fundamental studies. Our experiments confirmed the possibility of long-term culturing of cells maintaining trophoblast properties. The identity and purity of 4 trophoblast cultures free from contamination and retaining the properties of pure culture during long-term (>10 passages) culturing in vitro were confirmed.

Isolation and culturing of trophoblasts from human terminal placenta.

Trophoblast culture was derived from the islets of cell migrating from the fragments of placental microvilli. The isolated cells retained their cytotrophoblast phenotype longer (until passage 2-3) and then gained characteristics of proliferating mesenchymal cells. We conclude that changes in the morphology and co-expression of epithelial (cytokeratin 7) and mesenchymal (vimentin) markers attest to epithelial mesenchymal transition.

Pilot in vitro study of the parameters of artificial niche for osteogenic differentiation of human stromal stem cell pool.

The aim of this research is experimental investigation of the topography and evaluation of some parameters of artificial microterritories promoting osteogenic differentiation of stromal stem cells. A technique of short-term culturing of prenatal human lung stromal cells with fibroblastoid morphology on calcium phosphate substrates with known topography was used. Judging from secretory activity of the cell culture (osteocalcin, alkaline phosphatase), stromal stem cells directly interacting with calcium phosphate discs have advantage in manifestation of osteoblast-like functional activity in comparison with cells cultured on plastic. Rough surfaces of calcium phosphate discs stimulate the formation of spatial human fibroblastoid cell culture. The cells with positive reaction to acid phosphatase are located on spheroliths forming the relief of calcium phosphate coatings. The cells with positive reaction to alkaline phosphatase (marker of osteoblasts) populate hollows (niches) of the artificial surface. The niche for induction of osteogenic differentiation of human multipotent mesenchymal stem cells is apparently a structural and functional formation. It can be characterized by an index calculated as the ratio of the total area occupied by alkaline phosphatase-positive cells to the area of artificial surface occupied by one stained cell.

[Transcription factor YY1 participates in activation transcription of the human ribosomal protein L11 gene].

Ribosomal protein L11 plays important role in ribosome, being involved in several steps in protein synthesis and also activates p53-dependent cell cycle arrest. Changes in the rpL11 levels might be implicated in cell cycle control and carcinogenesis. Therefore, the mechanism of regulation of rpL11 expression has increasing importance. Article presents research results of interaction of promotor elements of gene HRPL11 with proteins of nuclear extracts of cells of a various cell origin. Use oligonucleotide competitors containing known transcription factor-binding sites, and also polyclonal antibodies has shown, that transcription factor YY1 participates in regulation of a transcription of gene HRPL11 in all investigated cellular lines. Our data obtained from comparison of protein binding profiles using nuclear extracts from rapidly growth cells, normal cell lines and serum deprivation repressed cell allows us to consider of transcription factor YY1 as activator of HRPL11 gene transcription.

Comparative analysis of reproduction of influenza virus strains in cell lines perspective for the creation of cultural vaccines grown on nutrient medium on the basis of rise flour protein hydrolysate.

We studied vaccine strains of influenza viruses A and B during their culturing in MDCK and Vero cells grown in Eagle's MEM medium and in a medium on the basis of enzyme hydrolysate of rise flour proteins with reduced (2%) content of fetal calf serum. Optimal conditions for cell culturing and reproduction of influenza virus strains in these cells were studied. Culturing of vaccine strains of influenza viruses in MDCK and Vero cells grown in nutrient media on the basis of rise flour protein hydrolysate yielded high infection titers, which suggests that this medium can be used for the development of cultural influenza vaccine.

The use of components of plant origin in the development of production technology for live cold-adapted cultural influenza vaccine.

The impact of culturing conditions (multiplicity of cell culture infection with influenza virus, composition of growth and maintenance nutrient media) for the efficiency of multiplication of cold-adapted reassortant vaccine strains of influenza A and B viruses was evaluated. Soybean hydrolysate protein-based biological additive to nutrient medium provided effective reproduction of influenza A virus in MDCK cells in the presence of 2 microg/ml trypsin. The use of soybean peptone-based stabilizer provided retention of infectious titer of influenza virus grown in MDCK culture after its lyophilization to a level of 8.5 lg EID50/ml.

Study of morphofunctional peculiarities of fibroblasts isolated from human vertebral growth plates during in vitro culturing.

We studied structural and functional peculiarities of growth zone chondroblasts isolated from human fetal spines on gestation week 12 and cultured in vitro over 4 passages. The morphology of chondroblasts of different differentiation degree was described. It was found that the population of chondroblasts had certain features determined by changes in the relative content of cells of different differentiation degree. The data suggest that culturing conditions affect cell differentiation and the possibility of using primary human chondroblast culture as the experimental model of differentiating human vertebral growth plate chondroblasts in vitro.

[Preclinical studies of the anticancer adenovirus cancerolysin preparation].

The anticancer drug Cancerolysin has been developed, by using the mutant Adel2 variant of human adenovirus serotype 5 designed at the State Research Center of Virology and Biotechnology. Cancerolysin possesses a high degree of replication activity for complementary cells 293 and p53-deficient tumor cells and, at the same time, has significant replication limitations in normal human cells. Preclinical studies of the drug on laboratory animals (mice, rabbits, guinea pigs) have demonstrated its harmlessness and safety. When stored at -40 and -70 degrees C, the drug showed no significant activity throughout the control observational period (1 year).

[Further development (MDCK) of live cold-adapted influenza vaccine: cultivation of vaccine strains in production fermenters].

Optimal conditions were developed for cultivating the cold-adapted reassortant live influenza vaccine (CARLIV) in MDCK cells, which were in their turn cultivated in fermenters with serum-free medium and microcarrier. The use of MDCK cells meets all national and WHO requirements to continuous cells used in the production of biological preparations. CARLIV cultivated under such conditions well preserve their ts-mutations and mutation, which entail substitutions of amino acids, in all CARLIV genome segments. Provided the cultivation conditions are optimal, the output of multivalent CARLIV in a 101 fermenter can reach 100000 doses.

[Development and certification of libraries of the MDCK continuous cell line for production of influenza vaccine].

Seeding and working banks of the continuous MDCK cell culture suitable for the production of cultured influenza vaccine were created and deposited at liquid nitrogen temperature at the "Vector" State Research Center of Virology and Biotechnology. The MDCK cell culture was shown to have morphology typical of the discussed cell line; it does not have any alien agents and is oncogenically safe; its enzimogram and karyotype are typical of the donor line; finally, its biological properties are stable during a long period of cultivation and its sensitivity to influenza virus is high, therefore, it can be recommended for the production of influenza vaccine. The continuous MDCK cell line was certified at Tarasevich Committee and was recommended by the MIBP Committee, Russia's Health Ministry, for its use as a substrate in the production of diagnostic and preventive immunoglobulins.

[Characteristics of continuous cell line 293 used for preparation of antineoplastic agent Cancerolysin].

Certification of continuous cell 293 culture used for cultivation of antineoplastic preparation Cancerolysin was carried out. The seeding and working banks of cells 293 were established and deposited for storage at the Vector Centre. The cells were certified in accordance with the WHO requirements. The cell 293 culture was shown to have high proliferative activity; morphology typical of the line; its karyotype and enzymogram are typical of human cells; the culture is not contaminated with bacteria, fungi, mycoplasms and viruses including oncogenic ones; it has high virus-producing activity; it preserves stability of all the biological properties in long-term cultivation. The seeding and working cell banks were recommended for the use in production of drugs for the treatment of oncologic patients.

[Structural and functional analysis of the human ribosomal protein L11 gene]. / Strukturno-funktsional'nyi analiz gena ribosomnogo belka L11 cheloveka.

The nucleotide sequence was established for the human ribosomal protein L11 gene (HRPL11). The gene (4583 bp) proved to consist of six exons and five introns. The structure of the HRPL11 promoter was shown to be typical for ribosomal protein genes of higher eukaryotes: the promoter lacks TATA or CAAT boxes and has a TATA-like element (ATAA, -24 ... -27) surrounded by GC-rich regions, the transcription start point is within an oligopyrimidine tract, and the 5'-untranslater region is short (26 bp). Proteins interacting with the HRPL11 promoter were identified by the electrophoretic mobility shift assay with oligonucleotide competitors containing known transcription factor-binding sites. The promoter regions were compared for the human genes for ribosomal proteins L11, L32, and S26.

[Morphology and formation of symplasts in cell cultures infected with measles virus]. / Osobennosti morfologii i formirovanie simplastov v kul'turakh kletok, zarazhennykh virusom kori.

The morphology and formation of giant cells were studied in diploid r-68 and MRC-5 cells in comparison with Vero cells at different times after inoculation with measles Leningrad-16 virus by electron and light microscopy and morphometry. The virions were released mainly from mononuclear cells and small syncytia. The nuclei were positioned centrally in the syncytia formed by diploid cells and at the periphery in Vero cell culture. The examined cultures were similar in terms of virus titers but differed by morphology, size and number of syncytia per unit of surface and time course of their formation. In comparison with Vero cells, in diploid r-68 and MRC-5 cells the maximal number involved in giant cells was 5 and 3.5 times lesser and the number of syncytia per surface unit 20 and 13 times lesser, respectively. Human r-68 diploid cell culture was characterized by the least number and size of syncytia over the entire course of experiment and longest life of the monolayer.

[Experimental study of hemostimulating properties of the tablet form of recombinant human erythropoetin]. / Gemostimuliruiushchie svoistva tabletirovannoi formy rekombinantnogo éritropoétina cheloveka v éksperimente.

Specific biological activity of the domestic preparation of recombinant human erythropoietin (RHEPO) in an original pill form was experimentally studied in vitro and in vivo. Administration of the RHEPO parent substance at a concentration of 0.5 10 U/ml significantly accelerated the growth of erythroid colony-forming cells (CFU-E) in the culture of intact nonadherent myelokaryocytes. The erythropoiesis-stimulating properties of the new RHEPO preparation are comparable with those of the well-known injection preparations. The drug action is related to increasing the erythroid cell content in the bone marrow and the reticulocyte and erythrocyte counts in the peripheral blood.

[Elaboration of live measles vaccine technology on human embryo lung diploid cell culture L-68]. / Razrabotka tekhnologii proizvodstva zhivoi korevoi vaktsiny na osnove kul'tury kletok émbriona cheloveka L-68.

Measles predominates among childhood droplet infections in many countries. Immunization of all human beings sensitive to this infection is the only radical measure in controlling measles. The quality of a vaccine is primarily determined by the properties of the virus strains and cell cultures and technology of production. Now live measles vaccine is produced in or country on the basis of fibroblasts from Japanese quail embryo. The production of live measles vaccine in the primary cell cultures has a number of drawbacks caused by the nonstandard pattern of the substrate and the probability of contamination. The use the certified human diploid cells deposited in liquid nitrogen in sufficient quantities is promising. The authors have elaborated a new technology of live measles vaccine production by using the Leningrad-16 virus strain on the basis of attested L-68 diploid cell culture from the human fetal lung. Experimental batches of vaccine were obtained and attested in accordance with the present requirements for immunobiological products.

[A new cell line from Heliothis armigera (Hubn.) pupa ovaries]. / Novaia liniia kletok iaichnikov kukolok khlopkovoi sovki Heliothis armigera (Hubn.)

The new continuous cells line derived from insect cells is perspective for the production of nuclear polyhedrosis viruses. The culture was prepared by fragmentation and trypsin treatment of the ovaries of Heliothis armigera (Hubn.) pupa with subsequent inoculation of viable cells in nutrient medium. By the present time the culture has undergone more than 100 passages in vitro and is characterized by all parameters required in a present-day passport.

[Morphological and karyologic analysis of primary and continuous lepidopteran cells]. / Morfologicheskii i kariologicheskii analiz pervichnykh i perevivaemykh kletok cheshuekrylykh nasekomykh.

Morphological basis of obtaining primary and continuous cell cultures from different tissues and organs of Lepidoptera has been developed. Peculiarities of transfer of primary cell cultures from these insects to continuous ones have been detected. Two new cell lines from Heliothis armigera pupa ovaries and from Agrothis segetum embryos were derived.

[Identification of insect cell lines]. / Identifikatsiia linii kletok nasekomykh.

Identification of 10 cell lines of 6 insect species received by the cell culture collection of Molecular Biology Institute from different research institutions of the USSR was carried out by karyological analysis and determinations of the spectrum of isoforms of the following enzymes: glucose-6-phosphate dehydrogenase (G-6-PDH), lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), and superoxide dismutase (SOD). Nine out of the 10 cell lines examined were shown to be identical to those of gypsy moth. Possible ways of cell contamination with other cells in long-term cultivation and preparation of new lines were determined.