Culture medium for pre-implantation of goat embryos in vitro

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to veterinary science. A culture medium for pre-implantation embryos contains DMEM medium, inactivated blood serum of 6-7-month nanny-goats with blood content of IGF1 and IGF2 making 54.73 and 231.50 nmol/l respectively, glucose, streptomycin and penicillin, in the following ratio, wt %: inactivated blood serum of 6-7-month nanny-goats - 15.0-20.0; glucose - 0.40-0.45; streptomycin - 0.004-0.005; penicillin - 0.003-0.0035; DMEM medium - the rest.

EFFECT: application of the presented culture medium for pre-implantation of embryos leads to prolonged storage stability of an unfrozen embryo, and also improves engraftment of previously implanted embryos when transplanted from donor goats to recipients.

1 tbl, 6 ex

 

The technical field to which the invention relates.

The invention relates to the field of veterinary medicine, in particular to the media for embryo culture goats in vitro, and can be used in biotechnology of reproduction of other mammals, such as cows, horses, sheep and so on.

The level of technology

There is a method of cultivation of the zygote and/or embryo to be added to the medium for the cultivation of zygotes and/or embryos, including the location of the zygote or embryo in a culture medium and culturing in the culture medium add an effective amount of at least one of antisense oligonucleotide length 17-30 NK, each of which is complementary to the mRNA of at least one of the following genes: gene inducers Aptos, such as HRK, FAS, FASL, BAX, Caspasa-3, genes, growth factors such as IGF1, genes, receptors, growth factors, such as IGF1R, genes regulating the rate of fragmentation of embryos, such as HLA-E, HLA-F, HLA-G genes of cellular stress, such as HSF-1, HSF-2.

In the way that the zygote or embryo previously subjected to cryopreservation.

In the way that the zygote or embryo after cultivation subjected to cryopreservation.

In the way that the culture medium contains at least one of the following salts: sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, feast is at sodium, sodium lactate.

In the method, at least one of the antisense oligonucleotides added to the culture medium, modified.

In the way the length of antisense oligonucleotide is 18-21 NC.

In the method, the antisense oligonucleotide or antisense oligonucleotide added to the culture medium before placing the zygote or embryo in it.

In the method, the antisense oligonucleotide or antisense oligonucleotide added to the culture medium after the zygote or embryo in it.

In the way different antisense oligonucleotides successively added to the culture medium after the zygote or embryo in it.

In the method of cultivation is performed in the period after fertilization in vitro until the expiration of six days.

The composition to be added to the environment of the cultivation of zygotes and/or embryos that includes one or more antisense oligonucleotide length 17-30 NK, each of which is complementary to the mRNA of at least one of the following genes: gene inducers Aptos, such as HRK, FAS, FASL, BAX, Caspasa-3, genes, growth factors such as IGF1, genes, receptors, growth factors, such as IGF1R, genes regulating the rate of fragmentation of embryos, such as HLA-E, HLA-F, HLA-G genes of cellular stress, such as HSF-1, HSF-2.

In the composition, at least one of antimuslim the x oligonucleotides modified.

In the composition of the antisense oligonucleotide selected from the group including duplex DNA, synthetic oligonucleotides, antisense RNA.

In the composition length of antisense oligonucleotide is 18-21 NC (see U.S. Pat. RU # 2281778, CL AC 35/54, C12N 5/08, OR 43/00, publ. 20.08.06 year).

Cultivation of pre-implantation mammalian embryos outside the body is an essential component when conducting experimental embryological studies, as well as embryologically work on cloning and obtaining transgenic animals. A significant percentage of children born through IVF program, which includes the cultivation and fertilization of oocytes in vitro. Technology manipulation of the embryos in veterinary medicine lags behind medical. The maximum percentage of engraftment when transplanted embryos in cows is 50%. For other animals, for which designed this way, settling down even lower. However, even in medicine, using the latest technologies the percentage of successful pregnancies in IVF is only 15-25% per cycle. Low settling down due to embryonic losses due to abnormalities in the development of embryos or inability to implantation. The needs of the cultured embryos and optimal conditions for their development in vitro has not been finally determined.

Recognition is about, that a breach of the implantation process, even if the pregnancy occurred, has many implications, such as pre-eclampsia, intrauterine growth restriction, preterm birth and placental abortion. It is obvious that the available methods for culturing oocytes and embryos in vitro may have adverse effects on embryo development, including and affecting his ability to implantation.

Therefore, topical search and create specific environments for cultivation of pre-implantation mammalian embryos. As for the goats, with all the actuality of the topic, the medium for culturing embryos of these animals have not been developed.

Known environment, which studied the possibility of using complex medium ham's F-10 containing amino acids, vitamins and other components. This is due to its wide use for fertilization in vitro of human oocyte and embryo culture of human and animal (Loutradis D.K., Kiessling A.A., Kallianidis K., Siskos K., Creatsas g, Michalas s, Aravantinos D. A preliminary trial of human zygote culture in Ham's F-10 without hypoxanthine. J Assist Reprod Genet 1993; 10: 4: 271-275). In particular, it is shown that in this environment the embryos of mice and humans are able to develop to the blastocyst stage, and to hatch from the pellucid zone (Shirley Century, Wortham E.J., J. Witmyer et al. Effects of human serum and plasma on development of mouse embryos in culture media. Fertil Steril 1985; 43: 1: 129-134; Kruge T.F, Stander F.S.H, Smith K., Lombard C.J. The development of one - and two-cell mouse embryos in the absense of human serum. S Afr Med J 1986; 70: 9: 542-543; Dandekar P.V., Glass, R.H. Development of two-cell mouse embryos in protein-free and protein-supplemented media. J in vitro Fertil Embryo Trans 1990; 7: 2: 107-113; Krivokharchenko A.S., Velanovich LI, Tatarinova L.V., Ryabykh V.P. Development of mouse embryos in vitro in the absence of protein depending on the number of embryos in the microvolume environment. Ontogeny 1993; 24: 6: 53-60).

However, the commercial environment of ham F-10, along with other components contains gipoksantin, which in living organisms is an intermediate product of the catabolism of purine bases. There are reports that gipoksantin can have a negative impact on the development of bicellular mouse embryos in vitro, blocking their development in the first divisions of crushing. Reduced fertilization of oocytes and increases the frequency of cytoplasmic fragmentation of embryos (Loutradis D.K., John D., Kiessling A.A. Hypoxanthine causes a 2-cell block in random-bred mouse embryos. Biol Reprod 1987; 37: 311-316; Downs, S.M., Dow M.P.D. Hypoxanthine-maintained two-cell block in mouse embryos: dependence on glucose and effect of hypoxanthine phosphoribosyltransferase inhibitors. Biol Reprod 1991; 44: 6: 1025-1039; Dienhart M.K., Downs, S.M. Cyclic AMP rerversal of hypoxantine-arrested preimplantation mouse embryos is EDTA-dependent. Zygote 1996; 4: 2: 129-137; Dienhart M.K., M.J. O'brien, Downs, S.M. Uptake and salvage of hypoxantine mediates developmental arrest in preimplantation mouse embryos. Biol Reprod 1997; 56: 1: 1-13). When this exception gipoksantina of environment ham F-10 improves the results of the cultivation of zygotes man is ka in this environment (Loutradis D.K., Kiessling A.A., Kallianidis K., Siskos K., Creatsas g, Michalas s, Aravantinos D. A preliminary trial of human zygote culture in Ham's F-10 without hypoxanthine. J Assist Reprod Genet 1993; 10:4:271-275).

In more simple than medium ham's F-10, and does not contain gipoksantin environment alpha-MEM embryos of mice better developed in vitro (Roudebush W.E., Often N.L., Butler WJ Alpha-minimum essential medium (MEM) enhances in vitro hatched blastocyst and cell number per embryo over Ham's F-10. J Assist Reprod Genet 1994; 11:4: 203-207).

Were also achieved good results in the cultivation of human embryos for the environment alpha-MEM with different protein supplements (Noda Y., Shiotani, M., Goto Y., Nakagama So, Umaoka Y., Mori T. Culture of human embryos in alpha modification of Eagl's medium under low oxygen tension and low illumination. Fertil Steril 1994; 62: 5: 1022-1027; Desai N. D. Kinzer, Loeb A., Goldfarb J. Use of synthetic serum substitute and alpha-minimum essential medium for the extended culture of human embryos to the blastocyst stage. Hum Reprod 1997; 12: 2: 328-335).

The cultivation of rabbit zygotes in a mixture of DMEM media is RPMI 1640 (1:1) without protein they have evolved significantly better than in protein-free medium 199, which contains much more features than the specified mix.

Thus, it is obvious that the complexity of the environment does not provide the best embryo development, the mix should contain components that are identical to the natural habitat of the embryo.

In the composition of nutrient media for cultivation of pre-implantation mammalian embryos are usually calf serum (FCS) or serum albumin. Adding protein to the media is 199 improved results, and the authors suggested that the protein may connects some "extra" components, eliminating, thus, their negative effect (E.W. Carney, Foote R.H. development of Improved rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium. J Reprod Fertil 1991; 91: 1: 113-123).

These "extra" for pre-implantation mammalian embryos components in addition to gipoksantina is, perhaps, nicotinamide, which is part of many complex commercial cultural environments. It specifies the operation, which was shown a negative effect of nicotinamide on in vitro mouse embryos (Tsai F.C., Gardner D.K. Nicotinamide, a component of complex cultire media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer. Fertil Steril 1994; 61: 2: 376-382).

However, still not all components of these additives accurately identified, and known for not always clarified their effect on the embryo.

In recent years, the attention of researchers is attracted insulin-like growth factors (IGF, insulin-like growth factor 1 and 2 single-chain polypeptides containing 70 and 67 amino acid residues, each with a molecular mass of 7000-8000 Yes. Insulin-like factors are named because of their ability to stimulate glucose uptake in muscle and adipose tissue is similar to insulin. The structure of IGF-1 and 2 are homologous to insulin, their synthesis is mainly (but not only) in the liver, stimulare the Xia somatotropic hormone and meals and are hormonal mediators of the action on the tissues of somatotropic hormone. The system of the insulin-like growth factors, their binding proteins and receptors involved in the processes associated with growth and development of the organism, maintaining the normal functioning of many body cells. In the blood circulating IGF associated with the protein. The residence time in the blood more than somatotropic hormone. Circulating IGF-1 increases insulin sensitivity. Researchers from Stanford University (USA) showed that the growth factor IGF-1 promotes the healing of damaged muscles by grasping the stem cells from the bloodstream.

(Alessandra Sacco, Regis Doyonnas, Mark A. LaBarge, Mark M. Hammer, Peggy Kraft, and Helen M. Blau, 2IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors. JCB. 2005. Volume 171, 483-492).

Newborns in the plasma are determined by the traces of the IGF, in the period of his childhood level graduale grows, reaching a maximum at the age of puberty. Also known facts increase IGF during pregnancy. There are reports that IGF-2 is secreted placental cells cytotrophoblast and promotes migration of these cells in the decidual lining of the uterus (Tietz Clinical guide to laboratory tests. 4-th ed. Ed. Wu A.N.B.-USA, W.B Sounders Company, 2006, 1798 p.; Dufour D. Clinical use of laboratory data: a practical guide. - Williams & Wilkins. - 1998 - 606 p.; LeRoith D. and Roberts Ch.T., The insulin-like growth factor system and cancer Cancer Letters. Vol.195, Issue 2. 2003, Pages 127-137; Metamaterial. Secrets of endocrinology. M.-St. Petersburg. Ed. Bean - Not the ski Dialect.

A known environment in which to improve the quality of the embryo and implantation process on Wednesday injected IGF-2, together with prothrombin, which act as antagonists as regulators ' ability cytotrophoblast be subject to migration or emigrational behavior. The composition of the medium (one of the variants) are:

0.0003 to 750 ng/ml IGF-II

0.01 to 50 [mu]g/ml plasminogen

0.01 to 50 [mu]g/ml urokinase plasminogen activator

Synthetic Serum Replacement (SSR<R>) (USA=Art Supplement)

Human serum albumin (HSA)

Glucose

Sodium pyruvate

Lactate

Potassium sulphate

Magnesium sulphate

Sodium chloride

Sodium hydrogen phosphate

Non-essential Amino Acids

L-glutamine

Taurine

Sodium bicarbonate-heat

HEPES

Streptomycin 50 mg/litre

Penicillin 50,000 IU/litre

Phenol Red

(See Compositions and methods for culturing embryos and oocytes, patent number: WO 2007012117, publication date: 2007-02-01, inventor: Roberts Claire Trelford (AU), applicant: Adelaide Res & Innovation pty (AU); Roberts Claire Trelford (AU)classification: - international: C12N 5/02; A61K 38/30; C12N 5/08; C12N 5/02; A61K 38/30; C12N 5/08; european: C12N 5/06 B2E; C12N 5/06 B4F, application number: WO 2006AU01042 20060727, priority number(s): AU 20050903997 20050727).

This method gives good results on embryo implantation and development of pregnancy, but it has drawbacks.

1. First of all, that added potent synthetic drugs. It is known that exceed physiological levels of IGF causes the proliferation of cancer cells, as well as premature aging (Anisimov, 1987; Mayerhofer et al., 1990, Rollo et al., 1996, Steger et al., 1993; Wolf et ah, 1993; Ward et al, 1994; Dilman, 1994; Miller et al., 1995; Meliska et al., 1997; Snibson et al., 1999; Brown-Borg, Rakozy, 2000). Research of the authors of the patent is limited only by the pregnancy, thus, the unknown consequences of such an environment.

2. In this environment, which is given as universal for all types of animals and humans, are not taken into account specific features which, as we know, are often the limiting factor to the use environment of the drug or method.

3. The composition of the medium is complex, with many components to it.

4. The method of cultivation is difficult and requires daily complex manipulation of the embryo.

5. The method requires the use of expensive drugs.

Closest to the technical essence and the achieved positive effect and adopted by the authors for the prototype is protein-free culture medium DMEM (Dulbecco''s modified Eagl''s medium), the composition of which is as follows:

L-arginine-HCl - 84,0 mg/l

L-cystine - 48,0 mg/l

L-glutamine - 584,0 mg/l

L-glycine - 30.0 mg/l

L-histidine - HCl·H2O - 42,0 mg/l

L-isoleucine - 105,0 mg/l

L-leucine - 105,0 mg/l

L-lysine-HCl - 146,0

L-methionine - 30.0 mg/l

L-phenylalanine - to 66.0 mg/l

L-serine - 42,0 mg/l

L-threonine - 95,0 mg/l

L-tryptophan - 16.0 mg/l

L-tyrosine - 72,0 mg/l

L-valine - 94,0 mg/l

D-Ca-patented - 4.0 mg/l

Choline chloride - 4.0 mg/l

Folic acid 4.0 mg/l

Inositol - 7.2 mg/l

Nicotinamid - 4.0 mg/l/p>

Pyridoxal - HCl - 4.0 mg/l

Riboflavin 0.4 mg/l

Thiamine - HCl - 4.0 mg/l

(See Radams. Methods cell culture for biochemists. - M.: Mir. - 1983. - 262 S.)

The disadvantage of this environment is the lack of natural components contained in blood serum and other biological environments that ensure the proliferation of embryonic cells, their ability to attach and respectiveiy:

1 - hormonal factors stimulating the growth of cells and their functions;

2 - factors of attachment and rasplastyvanija cells;

3 - transport proteins that carry hormones, minerals, lipids and enzymes.

The technical result that can be obtained using the present invention is to increase the retention of the embryo in unfrozen condition, improving its quality and increasing the level of engraftment of transplanted early embryos during their transplantation from goats donors to recipients.

The technical result is achieved by using media for culturing pre-implantation embryos goats in vitro, including DMEM, while it additionally contains inactivated serum 6-7-month-old goats in the blood which the content of IGF1 and IGF2, respectively 54, 73 and 231,50 nmol/l, glucose, streptomycin and penicillin in the following ratio of components, wt.%:

inactivated serumblood 6-7-month-old goats15-20glucose 0,400,45streptomycin0,004-0,005penicillin0,003-0,0035DMEMrest

The nature of the receiving environment for the cultivation of pre-implantation embryos goats in vitro is as follows. As the base used by commercial culture medium DMEM (Dulbecco''s modified Eagl''s medium). The composition of DMEM following:

L-arginine-HCl - 84,0 mg/l

L-cystine - 48,0 mg/l

L-glutamine - 584,0 mg/l

L-glycine - 30.0 mg/l

L-histidine - HCl·H2O - 42,0 mg/l

L-isoleucine - 105,0 mg/l

L-leucine - 105,0 mg/l

L-lysine-HCl - 146,0

L-methionine - 30.0 mg/l

L-phenylalanine - to 66.0 mg/l

L-serine - 42,0 mg/l

L-threonine - 95,0 mg/l

L-tryptophan - 16.0 mg/l

L-tyrosine - 72,0 mg/l

L-valine - 94,0 mg/l

D-Ca-patented - 4.0 mg/l

Choline chloride - 4.0 mg/l

Folic acid 4.0 mg/l

Inositol - 7.2 mg/l

Nicotinamid - 4.0 mg/l

Pyridoxal - HCl - 4.0 mg/l

Riboflavin 0.4 mg/l

Thiamine - HCl - 4.0 mg/l

In DMEM contribute 15-20% inactivated serum 6-7 mesoclimatic (in the blood which the content of IGF1 and IGF2, respectively 54, 73 and 231,50 nmol/l), 0,40-0,45% glucose, of 0.004-0.005% streptomycin and 0.003-0,0035% of penicillin. Serum inactivate the usual way by heating at 56°C for 30 minutes

Adding inactivated serum 6-7-month-old goats gives the following. At this age (stage of puberty) in animals, the maximum increase the level of insulin-like growth factors (IGF1, 2) (see table). in complex with proteins that stimulate the embryo's ability to implant. In addition, the serum contains proteins and enzymes, which allows not to use in the composition of the serum fetal calf (FCS) or serum albumin. Glucose in the medium also allows you to get as close to in vivo, when the early preimplantation embryo is surrounded by a rich medium containing glucose. Antibiotics are stabilizers and guarantee the safety of the environment. When cooking environment for the embryos of other species, it is recommended to take the serum of the species of animal for which the environment is designed.

The application environment provides a high percentage of developed embryos with a high capacity for implantation.

The ability to Mature in vitro blastocyst to hatch from the pellucid zone, and the ability hatched blastocyst to attach to the substrate, to form expressed inside the Yu-cell mass and allow the growth of cells of the trophectoderm are the criterion, allows you to judge the usefulness of these blastocysts, and hence on the quality of cultural media (Chida S., L. Mettler Screening test for mouse blastocysts as an index of the vitality of embryos. J in vitro Fertil Embryo Trans 1989; 6: 5: 310-312).

Obtaining embryos. Embryos intended for experiments was prepared as follows. Use goats sayanskoi breed. Multiple ovulation in goats induce the introduction on the 14th day of the sexual cycle 24 mg follicle-stimulating hormone (FSH) followed by injection of 125 µg of Astropan". Synchronization of the ovulatory process in the ovaries provide intravenous 800 units of human chorionic gonadotropin. Hunting is detected using goats probes. Goats artificially inseminated their sperm goats of the same breed. Leaching of embryos produced by the use of laparotomy on day 3 after insemination (day of insemination mistaken for a zero day). As the wash liquid used phosphate buffer solution.

Evaluation of embryos produced when viewing them under a microscope. Normal suitable for transplant embryos are characterized by a compact spherical shape, clear boundaries blastomeres with distinct cell membranes.

The implementation of the invention

Examples of specific receiving environment for culturing oocytes and compentation embryos goats in vitro.

PR is measures 1. The embryos are placed in plastic Petri dishes with media cultivation. Then 5-6 embryos are placed in glass capillaries. The volume of culture medium in the capillary should be about 20 μl. The capillaries are filled so that the ends remained the bubbles. Then the capillaries are placed in glass vials under a layer of paraffin oil. Bottles filled three-component gas mixture (5% CO2, 5% O2and 90% N2), tightly closed, immersed in a vessel with water and placed in a thermostat at 39°C. After 72 h of cultivation count the number of blastocysts, and after 96 h, the number of hatched blastocysts. Valuewise believe embryos, completely out of the pellucid zone.

The environment is prepared in the following ratio, wt.%:

inactivated serum
blood 3-5-month-old goats20,0
glucose0,45
streptomycin0,005
penicillin0,0035
DMEMrest

Results: after 72 h cultivation of the 2 embryos 19 embryos (82,6%) reached the blastocyst stage, after 96 h of cultivation hatched from the pellucid zone 16 blastocysts, or 69.6% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, gave a pronounced expansion of the glass cells of the trophectoderm.

Example 2. Carried out analogously to example 1, but the environment is prepared in the ratio of components, wt.%:

inactivated serum
blood 6-7-month-old goats20,0
glucose0,45
streptomycin0,005
penicillin0,0035
DMEMrest

Results: after 72 h of cultivation from 10 embryos all embryos (100%) reached the blastocyst stage. After 96 h of cultivation hatched from the pellucid zone 8 blastocysts, or 80% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, given the expansion of the glass cells of the trophectoderm andformed visible nodules of cells of the inner cell mass.

Example 3. Carried out analogously to example 1, but the environment is prepared in the ratio of components, wt.%:

inactivated serum
blood 6-7-month-old goats15,0
glucose0,40
streptomycin0,004
penicillin0,003
DMEMrest

After 72 h cultivation of 11 embryos all embryos (100%) reached the blastocyst stage. After 96 h of cultivation hatched from the pellucid zone 9 blastocysts, or 81.8% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, given the expansion of the glass cells of the trophectoderm and formed visible nodules of cells of the inner cell mass.

Example 4. Carried out analogously to example 1, but the environment is prepared in the ratio of components, wt.%:

inactivated serum
blood 6-7-month-old goats10,0
glucose0,40
streptomycin0,004
penicillin0,003
DMEMrest

Results: after 72 h of cultivation 12 10 embryos embryos (83,3%) reached the blastocyst stage. After 96 h of cultivation hatched from the pellucid zone 9 blastocysts, or 75% of the total number of embryos. The attachment of the blastocyst to the wall of the capillaries were noted.

Example 5. Carried out analogously to example 1, but the environment is prepared in the ratio of components, wt.%:

inactivated serum
blood 6-7-month-old goats25,0
glucose0,45
streptomycin0,005
penicillin0,0035
DMEMrest

Results: after 72 h of cultivation from 10 embryos 6 embryos (60%) reached the Tadei blastocyst. After 96 h of cultivation hatched from the pellucid zone 4 blastocysts, or 40% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, however, the expansion of the glass cells of the trophectoderm was poorly expressed.

Example 6. Carried out analogously to example 1, but the environment is prepared in the ratio of components, wt.%:

inactivated serum
blood 2-3-year-old goats20
glucose0,45
streptomycin0,005
penicillin0,0035
DMEMrest

Results: after 72 h cultivation of 19 15 embryos embryos (78,9%) reached the blastocyst stage. After 96 h of cultivation hatched from the pellucid zone 12 blastocysts, or 63.2% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, however, the expansion of the glass of the cells trafikdage what we were poorly expressed.

Thus, the optimal are the examples 2 and 3, which gave a good result, namely the result of the cultivation after 72 h all embryos (100%) reached the blastocyst stage. After 96 h of cultivation hatched from the pellucid zone 80-81,8% of the total number of embryos. All formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, given the expansion of the glass cells of the trophectoderm and formed visible nodules of cells of the inner cell mass. Thus, on the basis of the research results it was concluded that the addition to the environment for short-term storage and embryo culture serum 6-7-month-old goats, containing the maximum number of IGF1 and IGF2, improves the safety of the embryos and their ability to implantation.

Research content in the serum of goats are given in the table.

Table
The concentration of IGF1 and IGF 2 in the serum of goats (n=5)
The age of the goatsIGF1, nmol/lIGF2, nmol/l
1-7 Nam2,69,54
1-3 months12,2246,56
3-5 months19,1178,54
6-7 months54,73231,5
8-9 months16,967,6
1 year14,8255,58
3 years13,0446.81 / bbl

The test environment

The purpose of the experiment is to be set as a new medium for embryo culture goats in comparison with existing ones.

In the experiment using goats sayanskoi breed (n=10). Receipt and evaluation of embryos produced according to the described scheme.

After evaluation of the embryos randomly distributed into 4 groups. Embryos intended for cultivation in DMEM without protein, make 1 group (n=10), in DMEM medium containing 20% FCS ("Flow", England) - 2nd group (n=10); in the medium ham's F-10 ("Serva", Germany) - 3rd group; n=10), in DMEM ("Flow", UK) with 20% IGS - 4th group (n=12). 1-3 group was the control, group 4 - experimental. Embryos were transferred to plastic Petri dishes with media cultivation. Then 5-6 is aradise placed in glass capillaries. The volume of culture medium in the capillary should be about 20 μl. The capillaries are filled so that the ends remained the bubbles. Then the capillaries are placed in glass vials under a layer of paraffin oil. Bottles filled three-component gas mixture (5% CO25% of O2and 90% N2), tightly closed, immersed in a vessel with water and placed in a thermostat at 37°C. After 72 h of cultivation count the number of blastocysts, and after 96 h, the number of hatched blastocysts. Valuewise believe embryos, completely out of the pellucid zone.

Results: in the 1st group (DMEM without protein) after 72 h of cultivation 7 embryos (70%) reached the blastocyst stage, in the 2nd and 3rd groups (DMEM with 20% FCS and medium ham's F-10 with 8 embryos (80%) reached the blastocyst stage, in the 4th group (DMEM with 20% serum 6-7-month-old goats) all 12 embryos (100%) reached the blastocyst stage.

After 96 h in the 1st group hatched from the pellucid zone 5 blastocysts, or 50% of the total number of embryos in the 2-nd group - 6 blastocysts, or 60%, in the 3rd group - 5 blastocysts, or 50%, in the 4th group - 10 blastocysts, or 83.3%.

In the 4th group formed as a result of cultivation out of the pellucid zone blastocysts attached to the glass surface of the capillary, given the expansion of the glass cells of the trophectoderm and formed well what about the visible nodules of cells of the inner cell mass. In the 2nd group attachment was observed in 4 blastocysts of 5, but was less pronounced. In the 1st and 3rd groups of the hatched blastocyst attachment and growth of cells were noted.

Thus, from experiment, that bicellular embryos goats successfully develop to blastocyst and hatched from the pellucid zone in DMEM medium containing 20% IGS. The percentage of the development 23.3-33.3 per cent higher than in standard environments. Embryos cultured in DMEM medium containing 20% ISG, have a pronounced ability to attach to the substrate, to form a distinct inner cell mass and allow the growth of cells of the trophectoderm.

Thus, the claimed invention in comparison with the prototype and other known technical solutions has a number of advantages:

1. The application environment provides a high percentage of developed embryos with a high capacity for implantation.

2. The active substance in the environment - IGF - is a natural biological fluid in a balanced ratio with all the necessary nutrients for the embryo.

3. Taken into account specific features of animals for which the environment is designed.

4. The environment is simple in structure and easy to prepare.

6. The environment does not require the use of expensive drugs.

Media for cultivation of pre-implantation embryos goats in vitro, including the redu DMEM, characterized in that it further comprises inactivated serum 6-7 monthly goats in the blood which the content of IGF1 and IGF2, respectively 54,73 and 231,50 nmol/l, glucose, streptomycin and penicillin in the following ratio, wt.%:

inactivated serum 6-7
monthly goats15,0-20,0
glucose0,40-0,45
streptomycin0,004-0,005
penicillin0,003-0,0035
DMEMrest



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.

EFFECT: obtaining a NIH/3T3-174 cell line capable of producing the SURF-6 protein 10-20 times more than protein content in the initial unmodified fibroblasts when an inducing substance is added.

5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.

EFFECT: obtaining a NIH/3T3-174 cell line capable of producing the SURF-6 protein 10-20 times more than protein content in the initial unmodified fibroblasts when an inducing substance is added.

5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of producing three-dimensional matrices for tissue-like structures from animal cells. The method involves covalent bonding of histons with the surface of pre-activated biocompatible polymer microspheres made from crystalline dextran. The microspheres with covalently bonded histons are then deposited by centrifuging. Microspheres containing 160-200 mcg protein per 1.0 g are deposited on a substrate surface in amount of 0.5-1.0 mg per 1.0 cm2 and then dried at room temperature. Further, the substrate is washed with a solution at pH 7.5 to remove material which is not bonded to the substrate. The layer of microspheres obtained on the surface of the substrate on which cells are deposited is used as a base for obtaining tissue-like cell structures.

EFFECT: invention increases reliability of the structure and stability of the protein layer of the three-dimensional matrix, simplifies and reduces the cost of the method of producing three-dimensional matrices for tissue-like structures from animal cells.

6 cl, 11 dwg, 6 ex

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

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 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: biotechnologies.

SUBSTANCE: device generates one specific and selective signal, which has sinusoid configuration of wave and when applied to electrodes, coils or other field-generating devices, it results in generation of electric field, having amplitude of approximately 20 mV/cm in bone tissue at 60 kHz with approximately 50% cyclic mode. Specified electric field is specific and selective for activation of bone morphogenetic protein (BMP) gene expression measured by messenger RNA in specified bone tissue.

EFFECT: efficient treatment of bone fracture, prevention of fracture risk, slow knitting, non-unions, bone defects, arthrodesis of vertebrae, osteonecrosis and osteoporosis.

6 cl, 8 dwg, 1 ex

FIELD: microbiology.

SUBSTANCE: invention may be used for considerable reduction of variations in produce of recombinant proteins, which occur in process of cells cultivation with application of various batches of commercially available soy-bean hydrolysate. Medium is produced for cells cultivation, which does not contain animal proteins, by means of complementing of mediums, which do not contain animal proteins, with soy-bean hydrolysate and additionally with biogenic amine in the range of 1-18 mg/l. Produced medium is used to produce recombinant proteins by means of cultivation of according cells.

EFFECT: invention makes it possible to reduce contagiousness of product, and also to increase efficiency of growth and productivity of cells.

12 cl, 9 dwg, 12 ex

FIELD: pharmacology.

SUBSTANCE: nutrient medium for mammal cell cultivation contains balanced salt solution, a source of nutrients and growth-stimulating factors as a vegetable hydrolysate, amino acids and vitamins. As the vegetable hydrolysate, it contains an enzymatic hydrolysate of rice flour with with pH 6.2-6.7, content of amine nitrogen 2.2-2.8 wt %, residual nitrogen 3.0-4.2 wt % and/or soya flour with pH 6.2-6.7, content of amine nitrogen 2.9-3.5 wt %, residual nitrogen 5.6-6.6 wt % at the following content of components: enzymatic hydrolysate of rice and/or soya flour 3-20 g/l, amino acids 0.11-0.86 g/l, vitamins 0.55-22.5 mg/l, balanced salt solution to 1 l.

EFFECT: standardising and reduction in price of nutrient medium, specially for cultivation of MDCK and Vero cells used in manufacturing of culture influenza virus vaccines.

4 cl, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: during 10-14 days, a conditioned medium of stationary growth mesenchymal stem cells is introduced through rectum in microclysters in a dose 30-50 ml 1-3 times a day.

EFFECT: reduced hemorrhagic diathesis, erosion and ulcer ensured by waste products of mesenchymal stem cells.

3 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to field of biotechnology, medicine and cellular biology. Claimed is method of differentiation of monocytic pre-cursors of dendritic cells into immature dendritic cells, as well as method of producing mature dendritic cells using colony-stimulating factor of granulocytes - macrophages GM-CSF without other cytokines under conditions which prevent cell adhesion to reservoir.

EFFECT: invention can be applied in immunotherapy based on dendritic cells, for instance, for application in treatment of diseases, including cancer.

22 cl, 15 dwg, 1 tbl, 7 ex

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology and molecular biology. Proposed is a medium for redifferentiation of dedifferentiated chondrocyte, as well as a method of redifferentiating dedifferentiated chondrocyte into the initial chondrocyte.

EFFECT: obtaining transplantation materials used in transplantology, plastic surgery, as well as cartilage regenerative therapy.

7 cl, 15 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to versions of compositions used for separating cells, particularly blood cells, containing dextran, an antibody against glycophorin A, anti-CD 15 antibody, anti-CD 9 antibody, anti-CD 94 antibody, anti-CD 161 antibody and if necessary, other anti-CD antibodies and other components, necessary for dividing cells, particularly heparin and bivalent cations. An assembly and a method of separating cells using the assembly are invented. The invented compositions and method allow for agglutination of cells through identification of their surface, and can be used for identifying even rare cells with high output.

EFFECT: agglutination of cells through identification of their surface.

50 cl, 4 dwg, 19 tbl, 14 ex

FIELD: chemistry.

SUBSTANCE: proposed invention concerns biotechnology and nutrient media. The invention claims an additive produced by fermentative decomposition of fish meat for animal cell cultivation. The said additive allows for obtaining a nutrient medium for animal cell cultivation without mammal-originating components, and an albumen cultivated in such medium.

EFFECT: obtaining nutrient medium additive produced by fermentative decomposition of fish meat for animal cell cultivation.

5 cl, 9 dwg, 5 ex

FIELD: immunology, medicine.

SUBSTANCE: invention relates to pharmaceutical composition containing dendrite cells loaded with al least five testicle cancer antigen and containing no line-specific differentiating antigens. Aldo disclosed are isolated cell line expressing number of five testicle cancer antigen expressing no differentiating antigens and method for inducing of immune response in human by using of said composition.

EFFECT: new effective pharmaceutical composition for inducing of immune response.

56 cl, 16 dwg, 5 tbl, 7 ex

FIELD: medicine, endocrinology, pharmacy.

SUBSTANCE: invention proposes an agent eliciting antidiabetic activity. Agent represents nonglycosylated recombinant human granulocyte colony-stimulating factor. It differs from native preparation by absence of glycosyl group and the presence of additional methionine residue by its N-end. Effect of agent is associated with mobilization and migration of bone marrow mesenchymal stem cells, homing into pancreas. This results to reparation of insulin-producing activity of organ and normalization of the peripheral blood glucose level after monotherapy with recombinant human granulocyte colony-stimulating factor.

EFFECT: valuable medicinal property of agent.

3 tbl, 2 dwg, 1 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

Up!