RUSSIAN BRANCH PUBLICATIONS

       The CCA, the Russian Branch of the ETCS, and Institute of Cytology RAS (St. Petersburg) publish annually the Information Bulletin “Cell Cultures” (the author and editor-in-chief: M.S.Bogdanova; the editorial board: G.P.Pinaev, G.G.Poljanskaya, M.I.Blinova).

   The Bulletin publishes information on the main directions of studies on cell cultures, on activity of the CCA and the Russian Brunch of the ETCS, of scientific meetings, conferences, new materials and equipment for  cell cultivating.

    The CCA will present a brief content of each issue of the Information Bulletin. 

«CELL CULTURES”

Information Bulletin. Issue 19,  St. Petersburg, 2004, 43 p.

 

        The Main Directions of Studies on Cell Cultures in Institutions of  Russia

The object of cryobiology – the eukaryotic cell V.T.Kakpakov

The Vavilov Institute of Genetic of RAS

Cell Biotechnology and Tissue Engineering 

 THE USE OF THE CULTIVATED OF  THE NORMAL HUMAN FIBROBLASTS AND KERATINOCYTES FOR HEALING OF THE LEG ACUTE ULCERS

A.Ju. Lapin1,2, E.G.Topuzov1, M.A.Rubtzov2, N.V.Kalmykova3, M.I.Blinova3, G.P.Pinaev3

1Medical academy by I.I.Mechnikov,3 Institute of Cytology RAS, 2Hospital ¹ 18,

St-Petersburg, Russia

The data of the  healing  of the acute ulcers  of  the legs  by the   substitutive cell therapy method are presented.  The cell products (the skin derm and epidermis equivalents)  were prepared by using of  the cultivated of normal human keratinocytes and fibroblasts. The life of  the ulcer defects of the patients was up 5 months to 4 years. The process of a total epithelisation of an ulcer    in 14-22 days of 40 patients  took  place  19 patients; a decrease in size of  the ulcer to 70-80 % were observed with another 19 patients;  the ulcers of 2 patients were not changed. The analysis of  the obtained results  demonstrates about the perspective of the proposed method of the healing of the acute ulcers.

Cultivation of different origin osteogenic cells on bioactive gassceramics for further divelopment of bone tissue substitutive implants

N.S.Nikolaenko1, N.V.Tsupkina1, R.V.Deev2, I.L.Patokin3, L.N.Lisenok4, V.P.Orlov2, V.G.Gololobov2, G.P.Pinaev1

1Institute of Cytology RAS, St. Petersburg, nani@mail.cytspb.rssi.ru; 2Russian Military Medical     Academy,St. Petersburg; 3State Research Institute of Highly Pure Bioreagents, St. Petersburg; 4St. Petersburg State Technological Institute

The objective of this research was to study proliferation and osteogenic properties of newborn rat cranium bone cells, rat osteosarcoma ROS 17-2/8 cells and rabbit bone marrow stromal cells during their cultivation on bioactive glassceramics “Biosit” (“Elkor”, St. Petersburg). For this purpose cell proliferation indexes, alkaline phosphatase activities and cell monolayer characters were examined by hemocytometer, the Lowry method and scanning electron microscopy. According to our data implants for bone tissue substitution can be prepare on the base of material “Biosit” obtaining osteogenic cells from special parts of calvariae or bone marrow. 

Development of Problem… 

CHARACTERIZATION OF THE HUMAN EMBRYONIC STEM CELL LINES RECEIVED ON THE BASE OF CELL CULTURE COLLECTION OF THE INSTITUTE OF CYTOLOGY RAS

T.A. Krylova, N.S. Musorina, T.N. Goryachaya, N.N. Nikolsky, G.P. Pinaev, G.G. Poljanskaya

Institute of Cytology RAS, St. Petersburg; * e-mail: poljansk@mail.cytspb.rssi.ru

Long-term cultivation of human blastocyst-derived embryonic cells (hES) was performed. Several properties of hESs were examined to prove the state of continuous cell lines. These cells passed more 200 population doublings with the average population doubling time of 37.0+1.5h.  The isolated hESs refered to as HESC-1, HESC-2, HESC-3, HESC-4, cultivated on mitotically inactivated mouse embrionic fibroblasts (STO continuous cell line) formed multilayer colonies of various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription factor Oct4 which are characteristics of embryonic stem cells of different origin. Expression of hES specific cell surface antigenes (SSEA-3, SSEA-4, TRA-1-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal antibodies. An additional proves for species specificity of HESC lines is the lack of expression of mouse specific surface antigene SSEA1.  

Establishment and characterization of the human embryonic stem cell lines

M.V. Prizjkova, M.A. Lagarkova, A.V. Liakisheva, E.S. Revazova, N.V. Gnuchev, S.L. Kiselev

Institute of Gene Biology RAS, Moscow, s_kiselev@newmail.ru

We have established a number of human embryonic cell lines from blastocysts unacceptable for further use. Mouse feeder cells were used for cell lines establishment and propagation. Derived cell lines were characterized on the embryonic stem cells immuno markers, alkaline phosphatase, and telomerase activity. All cell lines demonstrated normal caryotype. At present all derived cell lines underwent more than 150 doublings. 

Technical Information

The nourishing environment C 46 for the invertebrate cell cultures. 

CHRONICLE

This section contains information on the  results of the conferences held in 2003 and on the conferences planned for 2004.

MISCELLANEOUS

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-          Online course “Introduction to cell cultivation”.

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-          The biomedical venture “The Cell culture center”.

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-          Information of the Cell Culture Association.

Instructions for authors

 The paper should not exceed 10 manuscript pages. The manuscript is to be accompanied by recommendation addressed to the Editorial Board of the Information Bulletin from the institution in which the work has been carried out. The paper should contain a brief resume, with the tittle of the work, names of the authors, institution, city.

The manuscript is to be accompanied by its electron variant: one file in MS Word for Windows. The file should have the font Arial Narrow, 13; interline space – 1.5; the indented line – 0.5 cm; the upper margin  – 2.5 cm, other margins – 2.1 cm each. The file may be e-mailed to the address of the Editorial Board.

Address of the Editorial Board

Dr. M.Bogdanova, Institute of Cytology RAS, Tikhoretsky Prosp. 4, St. Petersburg 194064, Russia.   Tel.: (812) 247-53-10,    fax: (812) 247-03-41, e-mail: msb@mail.cytspb.rssi.ru

 

«CELL CULTURES”

Information Bulletin. Issue 18,  St. Petersburg, 2003, 70 p.

 

        Section 1. «The Main Directions of Studies on Cell Cultures in Institutions of Russia»

 

 Usage of genes - activators of transcription  as a  new perspective approach in sea biotechnology.  N.A. Odintsova, S.V. Plotnikov, K.V. Kiselev, V.P. Bulgakov. Institute of Marine Biology, Far East Branch of RAS, Vladivostok, e-mail: inmarbio@mail.primorye.ru; Far-East State University.

                                                                 Abstract

    Many problems of biotechnology cannot be solved without continuous cell lines from marine invertebrates. Primary cell cultures from sea urchins have a low proliferative level that prevents the establishment of their long-term cultures. To increase expression levels of the cell growth regulatory genes of sea urchins and thus achieve enhanced cell growth, we used the transcriptional activator Gal4 gene, found earlier in yeast. Sea urchin embryos as well as embryo-derived cells were transformed with the Gal4 gene. This treatment was shown to result in formation of teratoma-like structures possessing neoplastic features and enhanced cell growth potential. After 2-month-cultivation, some pigments were found in cultivated cells from transfected embryos. The absorbtion  spectrum of these pigments was similar to that of a echinochrom -  biological active substance of a great therapeutic potential.

 

 Callusogenesis in anther culture of wheat: the cytological and histological analysis.

S.N.Abramov, O.A.Seldimirova, N.N.Kruglova.

Institute of Biology of Ufa Scientific Center of RAS, Ufa, Kruglova@anrb.ru

 

Abstract

    Cytological and histological analysis of developing androclynic morphogenic and non-morphogenic calli obtained in anther culture of wheat, was performed. The detailed light- and electron microscopy reveal sufficient difference between the morphogenic and non-morphogenic calli. The morphological variability of cells of morphogenic callus was established. The cells of certain zones of morphogenic callus are characterized as meristematic. The main peculiarities of the non-morphogenic callus are the absence of the meristematic character and gradual degradation of its cells.

 

Kidney cells proliferation induced by anti-DNA antibodies. D.A. Temnikov, Ò.À. Nevzorova, V.G. Vinter. Kazan State University, Kazan, Russia.

 

Abstract

The ability of pathogenic anti-DNA antibodies (Abs) in autoimmune diseases (systemic lupus erythematosus, SLE) to penetrate into the cells has already been proved. These antibodies can cause functional changes in the cells. Some antibodies can bind to the cell surface and lead to its lysis.  Other pathogenic Abs penetrate into the cell and bind with cytoplasmic or nuclear structures. However, the problem of how Abs affect cells still remains open.

In the present paper we carried out a comparative research of kidney cells proliferation in the presence of anti-DNA antibodies. The Abs were extracted from different sources: the SLE patients serum before and after prednisolone (the main drug in SLE therapy) treatment, and the donors’ serum. Several fractions of Abs were obtained from each serum, that differ in the anion-exchange cellulose binding. In the SLE serum we found the Abs fraction that can stimulate cell growth. Moreover, the short-term hormonal treatment resulted in the loss of this stimulating capability.

We suppose that Abs are one of the main organisms factors that cause proliferation of epithelial tissue in glomerulonephritis. Possible mechanisms of this influence are discussed.

 

 Section 2. «Cell Biotechnology and Tissue Engineering»

 

 On the question of xenotransplantation of culture from cells and tissues of endocrine organs.

I.S. Turchyn, I.I. Drozdovych. Coordination Center of Transplantation Health Ministry of Ukraine, Kyiv, dspatent@oldbank.com

Abstract

A new method for the treatment of endocrine disorders by transplantation of corresponding cell and organ cultures was developed. The biological properties of these cultures and their abilities for proliferation were studied and hormonal production were shown. They had an adequate reaction to tropic hormones of the pituitary. The methods of isolation and cultivation of cell and tissue cultures from the thyroid, parathyroid, adrenal glands, testes, pancreatic islands of human fetuses and newborn pigs were developed. The method prospects for clinical practice were revealed by the transplantation of corresponding cell and tissue cultures in diabetes mellitus, hypocorticoidism, hypothyroidism and hypogonadism.

 

 Smooth muscle cell culture in tissue engineering of heart valve transplants. V.S. Akatov, N.I.Rindina, V.V.Soloviev, R.M.Muratov, L.A.Bokeria.

Institute of Theoretical and Experimental Biophysics of Russian Academy of Science, Pushchino; Bakulev Center of Cardiovascular Surgery of Russian Academy of Medical Science, Moscow; e-mail: v-akatoff@mail.ru

Abstract

Ability of rat smooth muscle cell seeding on porcine aorta wall fragments aimed to reduce their calcification after implantation in rats was studied. The calcium deposits in aorta wall fragments seeded by the cells were shown to be significantly smaller with respect to unseeded fragments. The results obtained point to the efficiancy of seeding a heart valve transplant by smooth muscle cells taken from a recipient to suppress tissue calcification after the implantation.

 

Establishment and use of human myoblast cell lines for cell mediated therapy of Duchenne myodystrophy  and in other medical and biological assays. Terekhov S.M.1, Krokhina T.B.1, Egorov E.E.2, Kazimirchuk E.V.2, Makarenkov A.S.1,   Smirnova T.D.1, Shishkin S.S.11Research Center for  Medical Genetics, Moscow,  2Engelhardt Institute of Molecular Biology, Moscow. E-mail: Terekhov@nm.ru  

                                                        

Abstract

Basic features of human satellite muscle cells (SMC) isolation and mass cultivation were reviewed in this paper. The possibility of using myoblasts derived from SMC cultures of healthy donors for cell mediated therapy of Duchenne myodystrophy  was also discussed.  It was shown that sequentiall treatment of muscle tissue with Versen solution, then 0,1% trypsin solution and then 0,1% collagenase solution is a perfect way to isolate  SMC from muscle tissue. This allows to get a material, that contains about 80-90% of  SMC cells. The efficiency of this approach is about  300 000 SMC cells per 0,5 g of muscle tissue weight. The isolated SMC cells can be stored in liquid nitrogen without significantly loss of viability, if the cells were frozen immediately after isolation in F12 culture media with 20% embryonal calf serum and 10% DMSO. Cultivation of SMC in F12 culture media with some additions (epidermal growth factor, insulin, dexametazon, fetuin) provide a higherst intensity of cells proliferation. On the other hand, the DMEM culture media with addition of 2% embryonal calf serum induced differentiation of  SMC into  myoblasts most effectively for this. The content of carbon dioxide in air should be lower than 5%. The described approaches of cultivation provides an increase of  proliferation potential of muscle cells up to 40-45 passages. It is enough to producing of biomass  that consists of several  billions of cells. The muscle cells obtained from healthy donors were used for a cell mediated therapy of Duchenne myodystrophy. In general possibility of this therapeutic approach was shown, but it is inefficient for practical use. Some problems connected to the effectivity of cell mediated therapy  were  discussed in this paper. One of these problems is that the cell substitutional therapy requires immortal cells. The hTERT  transfected muscle cells have  telomerase activity but it does not lead to immortalization of the cells.

 

 Section 3. «Development of a Problem…»

 

 Effects on the cells of technogenic electromagnetic radiation and the protective devices. R.Ya. Podchernyaeva, T.M. Khizhnyakova, E.I. Melnichenko, G.R. Mikhailova. 

D.I. Ivanovsky Institute of Virology RAMS, Moskow, dkl@invir.msk.su

 

Abstract

Effect of the electromagnetic apparates on biological activity of human and animal cells and protective devices neutralizing the harmful effect of electromagnetic radiation were studied. It was demonstrated that protective devices produce a  protective effect providing variability and growth activity of the cells.

 

 Morphogenesis of embryonic echinoderm cells in culture. A. Koptyaeva

Institute of Marine Biology Russian Academy of Sciences Far East Branch, kann@mail.primorye.ru

 

Section 4. ”New Methods of Studies on Cell Cultures

 

  ZYMOGRAPHY - A FAST AND RATHER CHEAP METHOD OF DETECTING OF PROTEASES IN DIFFERENT BIOLOGICAL FLUIDS. I.V. Voronkina

Institute of Cytology, Russian Academy of Sciences, St-Petersburg, voron@mail.cytspb.rssi.ru

 

Abstract

Zymography is a quantitative and qualitative method of detecting proteases in different biological fluids - tissue extracts, wound fluids or conditioned cells mediums. The use of certain macromolecular proteins co-polymerized into electrophoresis gels allows fast and cheap detecting presence and activity of different proteases. With help of the zymography estimating protease activity and molecular weight could be done simultaneously. This paper discribes different modifications of the zymography assay for detecting various types of proteases and protease inhibitors. An example of the standard process of the gelatin zymography is given.

 

 Section 5. «Technical Information»

 The products of “New Brunswick Scientific” company (USA).

 

                             Section 6. «Chronicle»

 This section contains information on the  results of the conferences held in 2002 and on the conferences planned for 2003.

 

 Section 7. Miscellaneous     

-         Online course "Introduction to cell  cultivation."

-         Information of the Cell Culture Association.

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