Method for in vitro slow down of initial stages of differentiating embryonic stem cells of mammals except primates

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and cell biology. It has been established that sensitivity of mouse embryonic stem cells to clinostatting increases depending on cell development stages: from embryonic stem cell colonies to embryoid bodies. It has been shown that embryoid body clinostatting leads to slow down of the beginning of cardiomyocyte differentiation and significant reduction of the amount of cardiomyocytes cut. The invention can also be used in space medicine.

EFFECT: delay of neuronal differentiation on later differentiation stages.

2 cl, 8 dwg, 1 tbl, 1 ex

 

The invention relates to biotechnology, cell biology, and can be used to slow the initial stages of the development of the culture of embryonic stem cells in mammals except primates.

The level of technology

Evolutionary processes on Earth are carried out at a constant part of the gravitational field. It is known that the gravitational field contributes to the normal development and functioning of any living system on Earth. Modeling the effects of gravitational factor in laboratory conditions using a slow 2D-clinorotation - rotating cell culture relative to the gravity vector in a horizontal position at a constant speed, and the axis of rotation is perpendicular to the gravity vector, or ZV-clinorotation, when the position of the cell culture constantly randomisierte relative to the gravity vector [D. Moore, 1996; Jack J.W.A. van Loon, 2007].

A large number of experimental data performed on various types of cells, testify to their sensitivity to changes of the gravitational stimulation that cause structural changes and changes in the functional activity of the cells [Cogoli A., 1993; cosmonauts after long M.G., 2002; Crawford-Young ST., 2006; Borovkova LB, 2008].

It's very important to study the influence of clinorotation on the development, differe is a diverging pattern and regeneration of living organisms [Melekhov OP and others, 1976; Oganesyan, S. and others, 1980].

However, changes at the cellular level, what is happening in terms of real and simulated microgravity, do not allow at present to formulate General laws of this impact. Therefore, the question of the effect of gravity on the cell and the mechanisms of developing effects are very important.

The analysis of a large literature has shown that the question remains about the use of the method was shown that for culturing embryonic stem cells (ESCs) mammalian cells, which are at the very early stages of development and differentiation. ESCs are characterized by several very important properties: unlimited capacity for proliferation while maintaining a normal karyotype and the ability to differentiate into all known types of cells [Evans M.J., 1981; Guan, K. et al., 1999; Keller G., 2005]. Therefore, ESK and formed them embroidme body (EB) are an adequate model for studying the mechanisms regulating the initial stage of both spontaneous and induced various influences cell differentiation.

Cultivation of ESCs without substrate without addition of any factor that inhibits leukemia, leads to the formation of EB [Doetschman T.S. et al., 1985; Guan, K. et al., 1999], the cells spontaneously differentiate into derivatives of the three embryonic whether tcov. Adding a powerful specific inducers (e.g., retinoic acid or neurogenic growth factor, epidermal growth factor), you can receive targeted differentiation on cardiomyocytes or neuronal pathways [C. Strubing et al., 1995; Guan, K. et al., 1999]. The literature presents data on the impact at the initial stage of development ESK chemical and biological factors and almost no experimental results on the effects of mechanical factors on the ESC.

In the prior art the authors did not found documents clinostating cultures of mammalian cells at early stages of development, leading to a pronounced change in their development.

The essence of the claimed inventions

Conotation cell culture does not remove gravity, but the test sample is placed on clinostat, experiencing the impact of reduced gravity. In the experiments used a horizontal clinostat a rotation speed of 6 rpm, which corresponds to the acceleration of free fall 10-4g.

Education THIS reflects the entry of cells at the initial stage of differentiation, which can change under the influence of various physical, chemical and biological factors [Wobus A.M., et al. 1991; C. Strubing et al., 1995; Guan K. et al., 1999; Gordeeva OF and others, 2003]. Assessment of the ability to form cells of THIS was carried out in the period after impacts the Vija of clinorotation (72 h) in the following passages ESK.

Examples illustrating the invention

In this work, we used a mouse ESCs line R1. Cells cultured on a substrate of inactivated by mitomycin C (5 µl/ml) embryonic fibroblasts were subjected to action of clinorotation (6 rpm, 72 h). For constant stirring cultivation media used horizontal shaker (60 rpm) - dynamic control. Cultivation of ESCs (15-33 passages, 150-200 testlets) was carried out in CO2-incubator ("Sanyo, Japan) at 37°C and 5% CO2on the vials of 25 cm in the medium α-MEM (ICN, USA)containing 15% characterized fetal cow serum (FBS) ("NuSOAP, USA), in the amount adopted for such environments, 0.1 mm 2-mercaptoethanol, 2 mm L-glutamine (ICN, USA), and nonessential amino acids (ICN, USA), nucleosides ("Sigma", USA), vitamins (ICN, USA), in the amount adopted for such media, antibiotic gentamicin (20 μg/ml), glucose (ICN, USA) and 1 mm sodium pyruvate (ICN, USA).

Quantitative analysis photographed colonies and ET, as well as their morphometry (area, perimeter, maximum and minimum diameter) were carried out using the computer program SigmaScan Pro5 ("Sigma", USA).

To assess the ability to form EB cells after clinorotation were sown on Petri dishes (35 mm in diameter, 200 thousand cells) in α-MEM. After 3-4 days after seeding cells produced adscam formed STOR (suspension 5 μl, 5 fields).

EB were cultured in the clinorotation (72 h), passiria 600 bodies in the culture flasks of 25 cm2in the medium α-MEM (ICN, USA), containing all of the above supplements. Conditions was shown that ET did not differ from the conditions that were described for culturing ESCs at the stage of the colonies.

Subsequent differentiation of cells in vitro into cardiomyocytes was performed after 72 h was shown that THIS method of registering the appearance of declining clusters. Counting educated declining clusters was carried out every 24 h under a microscope and digital camera; making the video cuts.

The method was shown that with subsequent neuronal cell differentiation in vitro was performed otherwise. ESK line R1 (21-25 passages) in the amount of 100-200 Tyskie was passively on the bottle (9.1 cm2in the same environment α-MEM. Educated EB were clinostating (48 h) and subsequently with each vial (experience, static and dynamic controls) were sown on 4-hole plates (8 EB per well). 18 days later performed immunocytochemical staining with antibodies against the neuronal antigens.

Processing of the results of education declining clusters of cardiomyocytes and neurons, as well as the counting of apoptotic cells and cavities inside THIS was carried out on the basis of the conventional package of the statistical software Statistica 7.0 for Windows using nonparametric methods for comparing two independent between groups (criterion Mann-Whitney) and method for assessing the reliability of the shift in values an economic indicator (criterion Friedman).

Analysis of the results

As a result of counting educated FL (initial stage of differentiation of ESCs) in the period following the steps of the method was shown that it was discovered that generated THIS experience tended to decrease compared with the series of the UK.

On the basis of no change in the formation of colonies of HESC proved that the method was shown not to affect the formation of colonies of undifferentiated cells.

Conotation FLOOR for 72 h in vitro showed the presence of different morphology FLOOR in three samples (SC-static control, DK-dynamic control and clinostat) (figure 1). In particular, it is shown that clinestosterone THIS due to the constant rotation are supported in a suspension state, and most importantly, is how at the end of the impact visually, it was found that clinestosterone THIS differed more smooth and rounded shape (figure 1).

In the period aftereffect spent observing the advent of declining clusters of cardiomyocytes. Time reductions in all three groups continued for 14 days (figure 2). It was noted that in both test series the appearance of shrinking clusters occurred on the 3rd day after the end of the experiment, in contrast to the experience, which was Yavlena delay for 2 days. The results of statistical analysis showed significant differences in the time during which the reduced clusters of cardiomyocytes in the groups studied (figure 2). Thus, after long exposure was shown that ET (72 h) in vitro has been a significant delay in time of the beginning of the differentiation of ESCs in FL (p<0,01, according to the criterion of Friedman).

The maximum number of beats in FL for static and dynamic controls was observed on the 7th day after clinorotation (5-day reduction), and for clinostating FLOOR on the 11th (7-day reduction). The data of other researchers, describing the spontaneous differentiation of mouse ESCs in cardiomycytes direction, say the first twitch of clusters on the time period 2-3 days after attaching the FLOOR to the substrate [Guan et al., 1999; H. Wei et al., 2005], which is consistent with the results obtained in the control.

The evaluation of the total quantity of declining clusters in the period of their maximum, it was found a significant difference between all three samples (p<0,01 according to the criterion of Mann-Whitney). The results suggest that the number of cuts in the FLOOR, cultivated in the conditions of clinorotation (72 h), is reduced.

To confirm the presence of THIS cardiomyocytes was performed immunocytochemical staining of differencirovany the x ESC antibodies to troponin I - cell marker, which is known to be expressed in differentiated cardiomyocytes [Wei N., et al., 2005]. From figure 3 (arrows indicate stained cells) shows that the number of cardiomyocyte precursors to 18 days after clinorotation more in control, which confirms the weakening of spontaneous differentiation in the experience.

It is known that one of the directions of differentiation of mouse ESCs in vitro is differentiation into cells of the nervous system [Guan et al, 1998]. The assessment of the degree of spontaneous neuronal differentiation of ESCs after method influence of clinorotation was carried out using antibodies to specific proteins of nerve cells: MAR (MAP-microtubule associated proteins) and β-III tubulin. Previously it was shown that spontaneous differentiation into neuronal cells occurs in 15-20 days after insertion of the ET [Striibing S. et al., 1995]. The maximum number MAR-positive neurons differentiated from mouse ESCs in vitro, we detected approximately 18-20 days of cultivation [Fraichard, A., et al, 1995]. In connection with the foregoing, the study immunocytochemical staining of HESC-specific proteins ((3-III tubulin, MAR) was carried out in 20 days after the beginning of cultivation FL.

MAR-positive and β-III tubulin-positive neurons were found in all variants of the study (experience, static control IC and dynamic to ntrol - DK) (figure 4). Morphologically they were cells with short and long shoots. Quantitative analysis of the results showed that the number of β-III tubulin-positive neurons in the experience significantly higher compared with the series of IC and comparable with series DK (p<0.05 criterion Mann-Whitney) (table 1).

Table 1.
The number of neuronal cells, identified on the 20th day of specific antibodies to β-III tubulin and MAR (% from SC).
experienceSKDK
β-III tubulin-positive neurons364±195* n=19100±0 n=12411±58 n=10
MAR - positive neurons65±9*100±0122±95
n=16n=16n=16
* differences between experience and SC; p<0,05.
n - number of holes.

The detection clinostatic is utilized cultures of a large number of cells, painted on β-III tubulin, due to the fact that the differentiation of a substantial part of the ESC stopped at the stage of neuroblastoma, and the terminal stage of differentiation reached a significantly lower number of cells. This is evidenced by the number MAR-positive cells clinostating EB, which was significantly reduced compared with a series of SC. There was a trend to decrease MAR-positive cells clinostating ET compared with DKK (table 1).

Analysis of expression of a marker of neuronal differentiation - MAR suggests that the constant mixing environment of the cultivation of slightly activated, and the conditions of clinorotation (48 h) reduced spontaneous differentiation of cells. The obvious fact is that THIS longer was in a loose condition, and as a consequence, has been delayed further developments.

Thus, data showing a decrease in expression of the markers in the late stages of neuronal differentiation, consistent with the results obtained from the study of spontaneous differentiation of ESCs into cardiomyocytes, where it was shown a reduction in the number declining clusters in FL after cultivation in conditions of altered gravity. Detection in clinostating cultures of a large number of cells stained for β-III tubulin can be Holy is connected with the the differentiation of a substantial part of the ESC stopped at the stage of neuroblastoma, and the terminal stage of differentiation reached a significantly lower number of cells. The method was shown that creates conditions for a specific delay differentiation of ESCs in neuronal direction in relation to the late stages of this process.

Analysis of the half-sections of the FLOOR (100 slices) showed that in the experiment the cells of the inner layer are less tightly than in control, and they can detect fragmented nuclei (figure 5).

During embryonic development of the mammalian early signal of apoptosis is cavitation, i.e. when part of the cells of the embryonic ectoderm, going into apoptosis, forms the cavity due to the activation of autophagy [Coucouvanis and Martin, 1995; X. Qu et al, 2007]. In the experiments according to the invention, as can be seen from Fig.6, in THIS control in the Central part was located sufficiently larger voids (arrows) compared to THIS experience. And around them can be detected cells, the morphological features resembling epithelium.

Detected (Fig.7) significantly fewer apoptotic cells per 1 mm square cut of THIS experience compared to control (p<0,01; criterion Mann-Whitney; ni=24 (experience), n2=67 (control)). When this relative size is alosta experience significantly lower than that in control (p< 0.05; criterion Mann-Whitney; p1=24, p2=70) (Fig).

Thus, we can conclude that the absence of large voids in clinostating is associated with a slowing of the process of autophagy and morphogenesis in FL. These changes could be delayed differentiation, discovered by the authors after clinorotation.

Using the method was shown that, for the first time, it was found that the sensitivity of the ESK to clinostating increases depending on the stages of development of cells from HESC colonies formed to FL. First it is shown that conotation THIS leads to the delay of the beginning of cardiomyocytes differentiation, and to a significant decrease in the number of declining cardiomyocytes. Demonstrated that the rotation of the cell culture relative to the gravity vector leads to a significant reduction of cells in the late stages of neuronal differentiation.

Novelty

The claimed invention has novelty, as first established that the sensitivity of mouse ESCs to clinostating increases depending on the stages of development of cells from HESC colonies formed to FL. First it is shown that klimastationen THIS leads to the delay of the beginning of cardiomyocytes differentiation, and to a significant decrease in the number of declining cardiomyocytes. the first shows the latency of neuronal differentiation at later stages of differentiation.

Inventive step

The claimed invention involves an inventive step, as in the prior art has not previously been shown significant influence of clinorotation on the development of mouse ESCs in the early stages of development. In addition, this assumption is no way to drain the current level of technology. On the contrary, the methodology used in the previous studies, did not show any significant influence on the development of mouse ESCs in the early stages of development. Thus, the claimed invention makes a significant contribution to the existing level of technology.

Brief description of drawings

Figure 1 shows the appearance of the FLOOR when clinostating (zoom×200): SC - static control, DK - dynamic control, CL - experience.

Figure 2 shows the dynamics of the emergence of declining clusters of cardiomyocytes in FL after a stop of clinorotation: CL - experience, SK - static control, DK - dynamic control.

Figure 3 shows immunocytochemical staining of differentiated HESC antibodies to troponin I after 18 days after clinorotation And experience (×200); B - control (×200). The arrow indicates stained cells.

Figure 4 shows the color of the differentiated cells of THIS specific markers (×200): MAR: a - SK; - experience; - DK; β-III tubulin: g - SC; d - experience; e - DK.

Figure 5 pok who are half-sections of the FLOOR (thickness of 1-2 μm). The color of methylene blue-Azur-fuchsin: a - control; b - experience (zoom×400). Thick arrows are void and the nucleus of apoptotic cells. Thin arrows cells of the outer layer.

Figure 6 shows the half-sections of the FLOOR (thickness of 1-2 μm). The color of methylene blue-Azur-fuchsin (zoom. × 400): a - control; b - experience. Arrow - cavity cavitation.

7 shows the number of mouse ESCs, which entered into apoptosis, per 1 mm2square half-slice of THIS.

On Fig shows the relative size of the cavities on the half-cut FL.

Sources of information

1. Borovkova LB problems of gravitational biology of the cell // the Aerospace and environmental medicine. 2008. V.42(6). P.10-18.

2. Gordeeva OF, Manuilova Y.S., Dime I.A., Smirnov Y.A., Krasnikova POSTGRADUATE, Zinoviev RD, N. Khrushchov Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro. Oncogenesis. 2003. T. No. 3. S-182.

3. Guriev T.S., Dadashev O.A., Meleshko GI, Shepelev DEATH, Boris K., Szabo C. quail Embryonic development in weightlessness // Space biology and aerospace medicine. 1993. So no. S-76.

4. Melekhov OP, Shileiko L.V., Burlakova O.V. Conotation embryos of amphibians of different age // In Proc. of gravity and the body. 1976.

5. Meleshko GI, Shepelev DEATH, Guriev T.S., Bodea K., Szabo C. AMB the optional development of birds in zero gravity // Space biology and aerospace medicine. 1991. T 25. No. 1. Pp.37-39.

6. Oganesyan, SS, Gevorkian, R.A., Seminar T.S., eloian M.A. effect on the myocardium chicken embryo simulated gravitational overload // Space biology and aerospace medicine. 1980. T. No. 6. P.54-57.

7. Serova L. C. Weightlessness and adaptive capabilities mammals / M IPGM. 1996. S.

8. In cosmonauts after long-MG Molecular and cellular basis of the gravity sensitivity. / / Moscow. 2002. 104 C.

9. Cogoli A. The effect of hypogravity and hypergravity on cells of the immune system // J. Leukocyte Biol. 1993. V.54. P.259-268.

10. Coucouvanis, E. and Martin G.R. Signals for death and survival: a two-step mechanism for cavitations in the vertebrate embryo // Cell. 1995. V.83. P.297-287.

11. Crawford-Young, S. J. Effects of microgravity on cell cytoskeleton and embryogenesis // Int. J. Dev. Biol. 2006. V.50. P.183-191.

12. Doetschman T.C., Eistetter h, Katz m, Schmidt W, Kemler R. The in vitro sevelopment of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium // J. Embryol. Exp. Morphol. 1985. V.87. P.27-45.

13. Evans M.J., M.H. Kaufman Establishment in culture of pluripotential cells from mouse embryos // Nature. 1981. V.292. P.154-158. Fraichard, A., et al, 1995. Guan K., Rohwedel J., Wobus, A. M. Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro II Cytotechnology. 1999. V.30. P.211-226.

14. Jack J.W.A. van Loon. Some history and use of the random positioning machine, RPM, in gravity related research // Advances in Space Research.

15. 2007. No. 39. P.1161-1165. 15. Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine // Genes & Dev. 2005. V.19. P.1129-1155.

16. Moore D., Cogoli A. Gravitational and spase biology at the cellular level. In Biological and medical reserch in space // An overview of life sciences research in microgravity. Ed. by More D., Bie P., Oser H. Springer. 1996. P.1-107. 17. Qu X., Zou Z., Sun, Q., Luby-Phelps K., Cheng P., R.N. Hogan, C. Gilpin

17. Levine B. Autophagy gene-dependent clearance of apoptotic cells during embryonic development // Cell. 2007. V.128. P.931-946.

18. Striibing C, Ahnert-Hilger G., Jin, S., Wiedenmann Century, Hesheler J., Wobus A.M. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and exciatory neurons // Mech. Dev. 1995. V.53. P.275-287.

19. Wei H., Juhasz A., Li J., Tarasova E.S., K.R. Boheler Embrionic ste, cells and cardiomyocyte differentiation: phenotypic and molecular analyses // J. Cell. Mol. Med. 2005. V.9. No. 4. P.804-817.

20. Wobus A.M., Wallukat G., Hesheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+channel blockers // Differentiation. 1991. V.48. P.173-182.

1. A way to slow the initial stages of the differentiation culture of embryonic stem cells (ESCs) mammals except primates, in vitro and their subsequent development in cardiomyocytes ways, including the following stages:
15-33 passages, 150-200 testlets ESCs cultured on a substrate of inactivated embryonic fibroblasts, is subjected to the action of clinorotation at about 6./min for 72 h, the cultivation of ESCs is carried out in CO2-incubator at 37°C and 5% CO2on the vials 25 cm2in the medium α-MEM containing 15% characterized fetal cow serum (FBS), 0.1 mm 2-mercaptoethanol, 2 mm L-glutamine, nonessential amino acids, nucleosides, vitamins, antibiotic gentamicin (20 μg/ml), glucose and 1 mm sodium pyruvate;
then the cells for 3-4 su is. sown on Petri dishes in medium α-MEM for education embryonic bodies (EB);
the resulting ET passedout 600 bodies on the bottle 25 cm2and cultured under conditions identical to the terms of the cultivation of ESCs on stage colonies, exposing clinostating for 72 h at about 6./minutes

2. A way to slow the initial stages of the differentiation culture of embryonic stem cells (ESCs) mammals except primates, in vitro and their subsequent development by neuronal pathways, which includes the following steps:
21-25 passages, 100-200 testlets ESCs cultured on a substrate of inactivated embryonic fibroblasts, passedout vial 9.1 cm2the cultivation of ESCs is carried out in CO2-incubator at 37°C and 5% CO2in the medium α-MEM containing 15% characterized fetal cow serum (FBS), 0.1 mm 2-mercaptoethanol, 2 mm L-glutamine, nonessential amino acids, nucleosides, vitamins, antibiotic gentamicin (20 μg/ml), glucose and 1 mm sodium pyruvate, to the stage of education embryonic bodies (EB);
the resulting ET clinostatic at about 6./min for 48 h;
then on a 4-well plate were seeded on 8 EB per well for 18 days in medium α-MEM containing all necessary additives, in terms identical to the terms of the cultivation of ESCs at the stage of the colonies.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there is offered a method for preparing a soft tissue filler composition for injection to relief or treat skin damages caused by mechanical or physiological reasons, including the stages as follows: 1) digestion of autologous dermal tissue recovered from autologous skin of a patient by processing with pancreatine/EDTA solution, and cell isolation; 2) cultivation and proliferation of the recovered dermal cells by serum-free cultivation in vitro in a medium containing a growth factor and activation factor for preparing autologous cellular culture material of dermal nature containing dermal fibroblastic stem cells, dermal fibroblastic "transitional" dividing cells, dermal fibroblasts and collagen; 3) centrifugation of autologous cellular culture material of dermal nature for separation of autologous cellular sediment of dermal nature; and 4) slurrying of autologous cellular culture material of dermal nature in glucose solution for injection or any solution for injection to prepare suspension for injection. There is offered a composition prepared by specified method which contains 1×107 to 8×107 cells/ml of autologous cells of dermal origins and 10 to 100 mg/ml of collagen as an effective ingredient.

EFFECT: invention provides therapeutic effect over a short period of time and maintains it for a long time.

13 cl, 7 ex, 8 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

5 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

5 cl, 3 dwg, 2 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 7D8, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain) and a strain of hybrid animal cells Mus musculus L. 7H10 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain). Monoclonal antibodies 7D8 which are produced by the strain of hybrid animal cells Mus musculus L. 7D8 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7H10 which are produced by the strain of hybrid animal cells Mus musculus L. 7H10 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7D8 and antibodies 7D8 and 7H10 are used together in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain).

EFFECT: use of the invention increases reliability of enzyme immunoassay.

5 cl, 3 dwg,1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line ILG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The ILG cell line is deposited in the Special collection of cell cultures of vertebrates of the Russian collection of cell cultures under number RKKK (P) 697D. All cells of the ILG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line 31G has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The 31G cell line is deposited in the Special collection of cell cultures of vertebrates of the Russian collection of cell cultures under number RKKK (P) 698D. All cells of the 31G line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line IG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The IG cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 700D. All cells of the IG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line 26G has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The 26G cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 701D. All cells of the 26G line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining cell lines and can be used for immunotherapy and immunoprophylaxis in patients with malignant growths. The human melanoma cell line PG has stable culture, morphological and immunological characteristics and can secrete a human recombinant granulocytic-macrophagal colony-stimulating factor (GM-CSF). The PG cell line is deposited in the Special collection of cell cultures of cage vertebrates of the Russian collection of cell cultures under number RKKK (P) 702D. All cells of the PG line are characterised by stable secretion of GM-CSF stored after inactivation of cells through exposure to 100 Gy of ionising radiation which reliably prevents proliferation of the inactivated cells.

EFFECT: invention enables introduction of inactivated tumour cells into patients in order to stimulate antitumuor immunity.

FIELD: medicine.

SUBSTANCE: method involves growing tissue culture Rauwolfia serpentina Benth (Rauwolfia serpentina) K-27 strain in a nutrient medium of given composition in the presence of melaphen (melamine salt of bis (hydroxymethyl) phosphinic acid) of 10-17 passages at a concentration of melaphen 1·10-3 g/l. After that received tissue culture Rauwolfia serpentina Benth is transplanted to the nutrient medium of the same composition but containing very small concentrations of melaphen 1·10-10-1·10-15 g/l, mainly at 1·10-13 g/l and is grown in this medium for a passage not more than 50 days.

EFFECT: increase in the accumulation of alkaloids.

3 cl, 5 dwg, 2 tbl

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

FIELD: organic chemistry, natural compounds, medicine, oncology.

SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.

EFFECT: valuable medicinal properties of compositions.

43 cl, 53 tbl, 50 dwg, 44 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: cellular biology, medicine.

SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

6 cl, 13 dwg, 3 ex

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

Up!