Banks of cell cultures for biotechnology.

Seeding and working cell banks were created and stored in cell culture collection. The banks were certified in accordance with international and national requirements. Cultures of 293, MT-4, L-68, FECH-16-1, FECH-16-2, 4647, MDCK, CHO TK(-), and CHO pE cells were recommended by Medical Immunobiological Preparation Committee for the use in the production of medical immunobiological preparations. The stock is sufficient enough for supplying standard cell material for the production of medical immunobiological preparations over few decades.

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.

[4647-cell culture for preparation of recombinant bivaccine against smallpox and hepatitis B].

The seeding and working banks of a 4647-cell culture have been created. The 4647-cell culture in these banks has a high proliferative activity, as well as the morphology, typical of this line, and the karyotype and the enzymogram, which are characteristic for the cells of an African talapoin (Cercopithecus aethiops). The culture is not contaminated with bacteria, fungi, Mycoplasma, and viruses, including oncoviruses. The deposited 4647 cells have high viral productive properties for the accumulation of the recombinant virus strain b7,5S2-S vaccine and keep the stability of all biological properties during a long-term cultivation. The continuous 4647 cell line was tested at the L. A. Tarasevich State Institute of SK. The seeding and working banks of 4647-cell culture at passages 108 and 128 are recommended as a substrate for cultivation of the strain b7,5S2-S vaccinia, used to prepare a bivaccine against smallpox and hepatitis B.

Development of cell culture (MDCK) live cold-adapted (CA) attenuated influenza vaccine.

Optimal conditions are determined for growing cold-adapted reassortant strains of a live influenza vaccine in MDCK cell line cultivated in a fermenter with a serum-free medium and microcarriers. The studied MDCK cell line meet all national and WHO requirements for the finite cell lines used for the production of biological preparations. CA reassortant vaccine strains grown in such conditions which fully preserve its mutations and the mutations lead to amino acid substitution in all genome segments of the studied CA reassortants. Under optimal cultivation conditions, the output of a monovalent live CA influenza vaccine in a 10-l fermenter may reach 100,000 doses.

[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.

[Development of microencapsulating measles live vaccine]. / Razrabotka mikrokapsulirovannoi formy zhivoi korevoi vaktsiny.

Designing of non-injection methods of immunization against measles has recently turned into a topical issue. Development of mucosal vaccines ensuring the "entry gate" immunity, which is highly effective in airborne infection, is in the focus of attention. The authors developed a method of microencapsulating the viral particles into the matrix of pH-dependent polymers. Microencapsulated live measles vaccine shaped as 0.6-2.0 microm particles was obtained. The specific activity of measles virus in the drug was 3.36-4.31 log TCD50/0.5 ml. In subcutaneous immunization of guinea pigs with capsules, the best results were obtained in a single administration of vaccine based on ethylcrylate, sodium alginate/ chitosan and sodium slaginate/HMDA. In the intranasal administration of vaccine based on sodium alginate/spermin and sodium alginate/HMDA, there was a need in 2 and 3 stages of immunization.

[Morphology of the microencapsulated form of measles vaccine based on pH-dependent polymers]. / Morfologiia mikrokapsulirovannoi formy korevoi vaktsiny na osnove pH-zavisimykh polimerov.

Morphology of microparticles of the microencapsulated measles vaccine was studied by cryofractography, transmission electronic microscopy and by atomic force microscopy; co-polymers of polyacrylic acid and of sodium-alginate (spermidine) complexes were used as the matrix. The different-composition microcapsules had clear-cut borders and a certain range of sizes; but they were different in morphology, and their structures and densities varied identically with regard for a medium acidity, which is apparently preconditioned by some conformation-type alterations of matrix molecules. The studied preparations can, probably, protect the viral material in the stomach aggressive medium and release the material to ensure its contact with the intestine lymph tissue; thereof, they can be referred to as promising for further study of mucosal vaccines.

[Development of a live culture of cold-adapted reassortant influenza vaccines]. / Razrabotka kul'tural'noi zhivoi kholodoadaptirovannoi reassortantnoi grippozonoi vaktsiny.

Optimal conditions for the cultivation of the MDCK cell lines in the laboratory spinner or by using the Eagle-MEM with or without fetal serum were worked out. The cold-adapted reassortant vaccine strains of virus influenza A/H1N1, A/H3N2 and B are well replicated in the MDCK cells both in a monolayer and in the spinner by using the serum-free medium. A maximum virus titer depends on a multiplicity of infection used in a fetal medium and on the addition of trypsin. Under the optimal conditions, the titer of the studied cold-adapted reassortants, while using a serum-free medium, reaches as much as 10(9.0)-10(9.5) EID50/ml.

[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.

Approaches to development of microencapsulated form of the live measles vaccine.

Development of delivery of antigens and antigenic complexes using microcapsules or microgranules made of pH-dependent polymers is one of several high priority directions of modern vaccinology. These polymers should protect the virus from acid gastric medium, dissolve or swell readily in weakly alkaline intestinal medium, in no way decrease the specific activities of viral antigens, promote their penetration into intestinal mucosa, and possess adjuvant properties. The State Research Center Vector and "DELSI" are developing the technology for production of microencapsulated form of the live measles vaccine L-16 viruses for oral administration. The authors have so far succeeded in selecting and characterizing a number of polymers that are promising for microencapsulated vaccine and for testing of virus titers and immune response of experimental samples of a new vaccine in animals. Control of samples in guinea pigs demonstrated that the encapsulated measles virus retained its specific activity and capability for inducing immune response in experimental animals.

[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.

[Ultrastructure of human L-68 diploid cells, infected with rubella virus]. / Ul'trastruktura diploidnykh kletok cheloveka L-68, infitsirovannykh virusom krasnukhi.

A monolayer of human diploid cell culture L-68 (21st and 30th passages) was infected with a intermediate variant of rubella vaccine strain Orlov and studied for 8 days by light and electron microscopy. Virus reproduction was associated with slight cytopathic effect; virions were detected in low amounts inside vacuoles and cisterns of the lamellar complex, groups of virions were found in the cytoplasm in association with cytomembranes and outside cells. Cell cultures differed by the cytopathic effect of the virus on the monolayer: microfocuses were found in the 21st passage culture, while in the 30th passage culture the monolayer was diffusely damaged.

[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.

[Prospects of use human fetal fibroblasts in the treatment of various etiology wounds]. / Perspektivy ispol'zovaniia fetal'nykh fibroblastov cheloveka pri lechenii ran razlichnoi étiologii.

The investigations have demonstrated a high efficiency of the certified human fetal lung fibroblasts in the treatment of skin wounds of various etiology. For treatment, a fibroblast monolayer grown on a backing was applied to various types of wounds (burns frostbites, donor site suppurations) and in shin phlegmon. There was wound debrediment and rapid skin recovery. Employing the cells from the certified cell culture banks provides not only a biologically and genetic uniform material, but also makes it possible to standardize treatments which are useful for wide medical application, by delivering the standard cells to medical institutions which have no experience in culturing. To set up banks of cell cultures and to store them for a long time at a temperature of liquid nitrogen in adequate quantities of ampoules allow the cultured fibroblasts to be provided with if a large quantity of cells is required, which may be useful in emergencies.