Method of cardiomyocyte selection with intracellular mitochondria used as indicator

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly method of cardiomyocyte selection from cardiomyocyte-containing cell mix without genetic alteration incardiomyocytes. Method of cardiomyocyte content boost in cardiomyocyte-containing cell mix without genetic alteration. Method of cardiomyocyte obtainment without genetic alteration to cardiomyocytes. Method of cardiomyocyte content assessment in cardiomyocyte-containing cell mix.

EFFECT: possible efficient selection of cardiomyocytes from cardiomyocyte-containing cell mix without genetic alterations.

20 cl, 16 dwg, 14 ex

 

The technical FIELD

The present invention relates to a method for the selection of cardiomyocytes from the cell population derived from a whole heart, or from a population of stem cells, and how they are used.

PRIOR art

Because the cardiomyocyte loses the ability to proliferation in the adult organism, in the treatment of severe heart disease, such as myocardial infarction or cardiomyopathy, it is necessary to conduct a heart transplant. However, currently available as an insufficient number of donor hearts, there is an urgent need to develop a method of treatment other than heart transplantation. On the other hand, it is expected that the attraction producedex vivocardiomyocytes will be the most promising way to help patients who need a heart transplant.

Developed different ways of obtaining cardiomyocytes, such as a method using differentiated embryonic stem cells, the method of stimulation and differentiation of stem cells (somatic stem cells)isolated from a living organism, which are thought to be in the body, and so on. However, in this area the problem lies in the fact that the characteristic feature of stem cells is that the% is CE differentiation/stimulation of stem cells as a by-product is always evolving cells, other than cardiomyocytes, and that undifferentiated stem cell always remains even after the process of differentiation/stimulation. Thus, in this field believe that differentiated/induced cell population by itself cannot be used in the method of treatment. Thus, it is necessary to select the cardiomyocytes differentiated from/stimulated cell population in order to successfully achieve transplantation in man's heart.

To date in this area has not been messages about effective method of purification of cardiomyocytes, in addition to the method of purification of cardiomyocytes pre-injection of the marker gene in the genome of stem cells (FASEB J., 2000, 14: 2540-2548). However, because the change of the genome includes significant ethical problems and cause unexpected serious risk, including changes in the rate of malignant change, the change of the genome for use in humans is questionable.

In this area it is known that the demand of oxygen is relatively higher than the need of major tissues other than the heart, and that the content of mitochondria in the myocardium is also relatively higher than the concentration in other tissues (Am. J. Physiol., 1985, 248: R415-421). In addition, this area is well known that cardiome the CIT largely loses the ability to mitosis after differentiation and maturation of cells. However, previously it was not known that the experts in this field have attempted to take the cardiomyocytes using the above properties of cardiomyocytes. In addition, there have been reported a direct comparison of the transmembrane potential of mitochondria of cardiomyocytes with a transmembrane potential of mitochondria in other cell types, focusing on the transmembrane potential of mitochondria and on the selection of cardiomyocytes using the transmembrane potential of mitochondria as an indicator of cardiomyocytes.

[non-patent document 1] FASEB J., 2000, 14: 2540-2548

[non-patent document 2] Am. J. Physiol., 1985, 248: R415 421

Description of the INVENTION

The PROBLEM SOLVED by the INVENTION

The authors of the present invention has sought to address the task of developing a method for the selection of cardiomyocytes without genetic changes from a cell mixture derived from a whole heart, and from the cell mixture derived from cells that can differentiate into cardiomyocytes using different types of properties of cardiomyocytes that are not directly related to selection of cardiomyocytes.

WAYS of SOLVING the PROBLEM

The authors present invention has successfully solved the above problem based on the discovery that the cardiomyocyte contains a relatively higher number of mitochondria than any other type of the notches, and that mitochondria of cardiomyocytes possess relatively higher transmembrane potential than any other type of cells. Based on these discoveries, the authors present invention has developed a new method for selecting cardiomyocytes without genetic changes of cardiomyocytes, which includes the following stages: stage labeling of cell mixtures containing cardiomyocytes, using specific mitochondria of the labeling reagent and the stage of measuring the relative content of mitochondria in the cells and/or transmembrane potential of mitochondria.

Specifically, in the first embodiment, the present invention relates to a method for the selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes, based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

Thus, in the first embodiment, the present invention relates to

the method of selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes based on the relative content of mitochondria in the cells;

the method of selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes based also regarding the nutrient transmembrane potential of mitochondria of cells; or

the method of selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes based on the relative content of mitochondria in the cells and the transmembrane potential.

This variant implementation of the method according to the present invention is characterized by the stages of tagging containing the cardiomyocytes cell mixture mitochondrial indicator and measuring the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

In the context of this invention containing the cardiomyocytes mixture of cells may be a mixture of cells derived from a whole heart, or cell mixture derived from cells capable of differentiating into a cardiomyocyte.

In addition, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

In this embodiment, after the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator and at the stage of measuring the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells, the method according to the present invention may further include a stage of cultivation labeled cleto is in the absence of a mitochondrial indicator.

Moreover, mitochondrial indicator used in this embodiment of the present invention may be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In the second embodiment, the present invention relates to a method of increasing the content of cardiomyocytes in containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes, where the method includes the following stages:

(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(2) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of the cell.

In the context of the second variant of implementation of the present invention containing the cardiomyocytes mixture of cells may be a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes.

In addition, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of with Volovoi cells, cells predecessor and egg.

In the second embodiment, after stage (1) and before stage (2) the method according to the present invention may further include a stage of cultivation labeled cells in the absence of a mitochondrial indicator.

Moreover, mitochondrial indicator used in this embodiment of the present invention may be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In the third embodiment, the present invention relates to a method for producing cardiomyocytes without genetic changes of cardiomyocytes, where the method includes the following stages:

(1) the stage of differentiation and stimulation of the formation of cardiomyocytes from the cell with the capacity to differentiate into a cardiomyocyte with a receipt containing the cardiomyocytes cell mixture;

(2) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(3) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

In the context of Exte third alternative implementation of the present invention the cell, having the ability to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

In addition, after stage (2) and before stage (3) the method according to the present invention may further include a stage of cultivation labeled cells in the absence of a mitochondrial indicator.

Moreover, mitochondrial indicator used in this embodiment of the present invention may be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, the present invention M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In the fourth embodiment, the present invention relates to a method for content evaluation of cardiomyocytes in containing the cardiomyocytes mixture of cells, where the method includes the following stages:

(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(2) the stage of measuring the ratio of cardiomyocytes and academician based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

In the context of the fourth version of the OS is enforced containing the cardiomyocytes mixture of cells may be a mixture of differentiated cells, which is obtained from the cell mixture derived from a whole heart, or from cells with the capacity to differentiate into cardiomyocytes.

In addition, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

Moreover, mitochondrial indicator used in this embodiment of the present invention may be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

As indicated above, in one embodiment, the present invention relates to a method for the selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

The relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells can be measured by marking containing the cardiomyocytes cell mixture mitochondrial indicator.

As used here, the term "containing ka is the cardiomyocytes mixture of cells" means a mixture of cells of all types, consisting of cardiomyocytes and other cell types. For example, "containing the cardiomyocytes mixture of cells" include, but are not limited to, a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into a cardiomyocyte. As used here, the term "mixture of cells derived from a whole heart" means a mixture of cells, consisting of cardiomyocytes, endothelial cells, stromal cells, smooth muscle cells, and so on, which is obtained by enzymatic treatment of homogeneous or heterogeneous cardiac tissue (heart) using different enzymes. In addition, as used here, the term "mixture of cells derived from the cells with the capacity to differentiate into a cardiomyocyte," means a mixture of cells, consisting of cardiomyocytes, academician, undifferentiated cells, neuronal cells and epithelial cells, which is obtained by culturing cells with the capacity to differentiate into cardiomyocytes (e.g., stem cells such as embryonic stem cells and somatic stem cells, precursor cells and oocytes, such as a fertilized egg and a clone of somatic cells), stimulation of cell differentiation of the cells having an ability to differentiate the I in cardiomyocytes.

The content of mitochondria in the cells and the transmembrane potential of mitochondria can be quantified by intracellular labeling of mitochondria mitochondrial indicator. However, the absolute value obtained by the mitochondrial indicator, which reflects the content of mitochondria transmembrane potential of mitochondria varies depending on the stage of maturation of cardiomyocytes subjected to analysis, and exposure conditions of the label, such as label type and time of exposure of the label. Thus, an important feature of the present invention is not an absolute value obtained using mitochondrial indicator signal, and the ratio between the amount received by the mitochondrial indicator signal for cardiomyocytes and signal for academician. In accordance with the present invention it is possible to select cells expressing relatively high fluorescence intensity, such as cardiomyocytes. First, on the basis of a preliminary experiment using a sample containing the cardiomyocytes cell mixture, which essentially can be used in accordance with the present invention, the determination of desired populations of cardiomyocytes (i.e. the ratio between the content of mitochondria and/or degree of t is incomprendido potential of the mitochondria of cardiomyocytes) is conducted properly in accordance with the purposes of this invention. Specifically, in the preliminary experiment based on the values of the content of mitochondria and/or extent of transmembrane potential of mitochondria used as indicators, the value obtained through the mitochondrial signal indicator cells intended for selection are classified into several groups. Cells, which shows a relatively high content of intracellular mitochondria, and cells, which shows a relatively high transmembrane potential of mitochondria, must be selected on the basis of the contents of the mitochondria and/or extent of transmembrane potential of mitochondria used as indicators.

In accordance with the present invention, the term "mitochondrial indicator" means, but is not limited to, a substance, such as a substance that can specific labelling of mitochondria in living cells and can display the content of mitochondria, a substance that can specific labelling of mitochondria in living cells and can display the transmembrane potential of mitochondria, or the substance that can specific labelling of mitochondria in living cells and can display the content of mitochondria, and the transmembrane potential of mitochondria. For example, "mitochondrial indicator" includes, but is not limited to, (1) vases is in, having a property to cause fluorescent emission and possessing the ability to bind the structural components of the mitochondria (e.g., a protein, a lipid, a sugar chain, a nucleic acid or a metabolite, etc.); (2) a substance having a property to cause fluorescent emission and built in mitochondria under the action of the transmembrane potential of mitochondria; (3) a substance that is converted into the form of a substance having a property to cause fluorescent emission under the action of the structural components of the mitochondria; or (4) a substance that loses its ability to diffuse out of the mitochondria outwards under the action of the structural components of the mitochondria.

In accordance with the present invention as illustrative of the mitochondrial indicator can be used mitochondrial indicator, such as, but not limited to, A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, S7563, T639, T668, T669 or T3168 (product numbers for connections: all available Molecular Probes); more preferably M7514, M7510, M7511, M7512, M7513, M22425, M22426, T668, R302 or T3168; most preferably T668, R302, M7514 or T3168. The chemical structure described above mitochondrial indicators used in accordance with the present invention, is presented below:

[Chem 1]

Structure A1372/p>

Molecular formula:C26H38BrN3

Molecular weight:472,51

CAS number:75168-11-5

Name:Acridine, 3,6-bis(dimethylamino)-10-nonyl-, bromide

[Chem 2]

Structure D273

Molecular formula:C29H37IN2O2

Molecular weight:572,53

CAS number:53213-82-4

Name:benzoxazolyl, 3-hexyl-2-(3-(3-hexyl-2(3H)-benzoxazolinone)-1-propenyl)-, iodide

[Chem 3]

Structure D288

Molecular formula:C16H19IN2

Molecular weight:366,24

CAS number:959-81-9

Name:pyridine, 4-(2-(4-(dimethylamino)phenyl)ethynyl)-1-methyl -, iodide

[Chem 4]

Structure D308

Molecular formula:C16H19IN2

Molecular weight:366,24

CAS number:2156-29-8

Name:pyridine, 2-(2-(4-(dimethylamino)phenyl)ethynyl)-1-methyl -, iodide

[Chem 5]

Structure D378

Molecular formula:C31H41N2O2I

Molecular weight:600,58

CAS number:there is no information

Name:there is no information

[Chem 6]

Structure D426

Molecular formula:C17H21N 2

Molecular weight:380,27

CAS number:3785-01-1

Name:pyridine, 2-(2-(4-dimethylamino)phenyl)ethynyl)-1-ethyl iodide

[Chem 7]

Structure D632

Molecular formula:C21H18N2O3

Molecular weight:346,38

CAS number:109244-58-8

Name:benzoic acid, 2-(3,6-diamino-9H-xanthene-9-yl)-, methyl ester

[Chem 8]

Structure D633

Molecular formula:C28H32N2O3

Molecular weight:444,57

CAS number:there is no information

Name:there is no information

[Chem 9]

Structure D22421

Molecular formula:C27H21IN2O3

Molecular weight:532,38

CAS number:there is no information

Name:there is no information

[Chem 10]

Structure D23806

INGREDIENT a: dihydrorhodamine 123

Molecular formula:C21H18N2O3

Molecular weight:346,38

CAS number:109244-58-8

Name:benzoic acid, 2-(3,6-diamino-9H-xanthene-9-yl)-, methyl ester

[Chem 11]

Structure L6868

Molecular formula:C28H22N4O6

Molecules of the RNA weight: 510,50

CAS number:22103-92-0

Name:9,9'-backriding, 10,10'-dimethyl-

[Chem 12]

Structure M7502

Molecular formula:C34H30Cl3N3O

Molecular weight:602,99

CAS number:there is no information

Name:there is no information

[Chem 13]

Structure M7510

Molecular formula:C24H24Cl2N2O

Molecular weight:427,37

CAS number:there is no information

Name:there is no information

[Chem 14]

Structure M7511

Molecular formula:C24H25ClN2O

Molecular weight:392,93

CAS number:there is no information

Name:there is no information

[Chem 15]

Structure M7512

Molecular formula:C32H32Cl2N2O

Molecular weight:531,52

CAS number:167095-09-2

Name:1H,5H,11H,15H-xanthene-[2,3,4-ij:5,6,7-I j']-dainolite-18-s, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-, chloride

[Chem 16]

Structure M7513

Molecular formula:C32H33ClN2O

Molecular weight:497,08

CAS number:167095-08-1

Name:1H,5H,9H,11H,15H-xanthene-[2,3,4-ij:5,6,7-I j']-dainolite, 9-[4-(HL is rmutil)phenyl]-2,3,6,7,12,13,16,17-octahydro-

[Chem 17]

Structure M7514

Molecular formula:C34H28Cl5N3O

Molecular weight:671,88

CAS number:201860-17-5

Name:benzoxazole, 2-[3-[5,6-dichloro-1,3-bis[[4-(chloromethyl)phenyl]methyl]-1,3-dihydro-2H-benzimidazole-2-ilidene]-1-propenyl]-3-methyl-, chloride

[Chem 18]

Structure M22422

Molecular formula:C35H33ClF6N2O

Molecular weight:647,10

CAS number:there is no information

Name:there is no information

[Chem 19]

Structure M22423

Molecular formula:C26H26ClN3O5

Molecular weight:495,96

CAS number:137993-41-0

Name:1H,5H,11H,15H-xanthene[2,3,4-ij:5,6,7-I j']-dainolite-18-s, 9-cyano-2,3,6,7,12,13,16,17-octahydro-, perchlorate

[Chem 20]

Structure M22425

Molecular formula:C39H36Cl5N3

Molecular weight:724,00

CAS number:there is no information

Name:there is no information

[Chem 21]

Structure M22426

Molecular formula:C34H36Cl2N2

Molecular weight:543,58

CAS number:there is no information

Name:there are no data

[Chem 22]

Structure R302

Molecular formula:C21H17ClN2O3

Molecular weight:380,83

CAS number:62669-70-9

Name:tsantili, 3,6-diamino-9-(2-(methoxycarbonyl)phenyl, chloride

[Chem 23]

Structure R634

Molecular formula:C28H31ClN2O3

Molecular weight:479,02

CAS number:989-38-8

Name:tsantili, 9-(2-(etoxycarbonyl)phenyl)-3,6-bis(ethylamino)-2,7-dimethyl, chloride

[Chem 24]

Structure R648

Molecular formula:C34H43ClN2O7

Molecular weight:627,18

CAS number:there is no information

Name:there is no information

[Chem 25]

Structure R14060

Molecular formula:C23H19F5N2O

Molecular weight:434,41

CAS number:there is no information

Name:there is no information

[Chem 26]

Structure R22420

Molecular formula:C21H17ClN2O3

Molecular weight:380,83

CAS number:62669-70-9

Name:tsantili, 3,6-diamino-9-(2-(methoxycarbonyl)phenyl, chloride

[Chem 27]

the structure T639

Molecular formula:C23H23N2OCl

Molecular weight:378,90

CAS number:6837-70-3

Name:tsantili, 3,6-bis(dimethylamino)-9-phenyl, chloride

[Chem 28]

Structure T668

Molecular formula:C25H25ClN2O7

Molecular weight:500,93

CAS number:115532-50-8

Name:tsantili, 3,6-bis(dimethylamino)-9-(2-(methoxycarbonyl)phenyl)-, perchlorate

[Chem 29]

Structure T669

Molecular formula:C26H27ClN2O7

Molecular weight:514,96

CAS number:115532-52-0

Name:tsantili, 3,6-bis(dimethylamino)-9-[2-(etoxycarbonyl)phenyl]-, perchlorate

[Chem 30]

Structure T3168

Molecular formula:C25H27Cl4IN4

Molecular weight:652,23

CAS number:47729-63-5

Name:1H-benzimidazole, 5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazole-2-ilidene)-1-propenyl]-1,3-diethyl-, iodide, (E)-

In addition, as a mitochondrial indicator can also be used S7563, S7567 and S7585 (product numbers for connections: all available Molecular Probes).

In this area it is known that each mitochondrial in icator has a certain wavelength excitation and causes the fluorescent radiation of a certain wavelength. For example, M7514 causes fluorescent emission 516 nm, emitted at a wavelength of excitation 490 nm, and T3168 causes fluorescent emission 590 nm, emitted at a wavelength of excitation 535 nm.

As described above, the cardiomyocyte shows higher fluorescence intensity than other types of cells when tagging containing the cardiomyocytes cell mixture mitochondrial indicator, because the cardiomyocyte has a relatively higher content of intracellular mitochondria and has a relatively higher transmembrane potential of mitochondria than other types of cells. First, in accordance with the present invention, the content of intracellular mitochondria and/or transmembrane potential of mitochondria measured for each cell, which is labeled in a mixture of cells containing the cardiomyocytes. Next, cells that have been shown higher fluorescence intensity, defined as the cardiomyocytes. Based on the measured content of mitochondria and/or transmembrane potential of mitochondria in cardiomyocytes isolated in accordance with the following: cell population with a relatively high content of mitochondria; cell population containing mitochondria with relatively high transmembrane potential; or cell population comprising cells having a relatively high content of mitochondria and relatively high transmembrane potential.

For example, if containing the cardiomyocytes mixture of cells machina fluorescent mitochondrial indicators c described above fluorescence, the cardiomyocytes (which shows the relatively high intensity of fluorescence due to the relatively high content of mitochondria and mitochondria, showing relatively high transmembrane potential) can be separated from other non-cardiomyocytes types of cells (i.e. cells which exhibit relatively high intensity of fluorescence-based label with mitochondrial indicators, due to the relatively low content of mitochondria or mitochondria, showing relatively low transmembrane potential) using a cell sorting device. In the sorting of cells can be selected viable cardiomyocytes without genetic changes of cardiomyocytes. Cell sorters used in the present invention may not be limited to the specific device, provided that they can be sorted viable labeled fluorescent-labeled cells. For example, as a concrete device for sorting cells according to the present invention can be used in cell sorters with activated fluorescence (FACS (registered trademark); BD, Franklin Lakes, NJ USA)and other devices for sorting cells (supplied by Beckman, Coulter, Cytomation, and so on).

Cardiomyocytes can be selected directly after labeling containing the cardiomyocytes cell mixture mitochondrial indicator. However, if you want to find the cardiomyocytes of containing the cardiomyocytes cell mixture more accurately, cardiomyocytes can be selected after labeling cells mitochondrial indicator, followed by the cultivation of labeled cells in the absence of a mitochondrial indicator. In the process of cultivation of labeled cells in the absence of a mitochondrial indicator after tagging mitochondrial indicator of the content of mitochondrial indicator, located in the same cell decreases as the cell undergoes cell division. Thus, over time culturing proliferating cells of the cellular mitochondrial indicator is reduced and the fluorescence intensity also decreases. On the other hand, since the cardiomyocyte is defined as the cell loses the ability to mitosis, or cell at a significantly reduced level of ability to mitosis, even after a long period of time cultivating the content of mitochondrial indicator, located in a separate cage, decreases more slowly than in other types of cells, and thus can be kept relatively higher the intensity of fluorescence. Thus, due to differences in the intensity of labelling between cardiomyocytes and academician, cardiomyocytes can be distinguished more accurately after tagging containing the cardiomyocytes cell mixture mitochondrial indicator, followed by additional culturing cells in the absence of a mitochondrial indicator within a certain period of time, more specifically in a few days.

In addition, in the second embodiment, the present invention relates to a method of increasing the content of cardiomyocytes in containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes, where the method includes the following stages:

(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(2) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

In the context of this case for containing the cardiomyocytes mixture of cells may be a mixture of cells derived from a whole heart or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes. As used here, the term "mixture of cells derived from a whole heart" means a mixture of cardiolite the s and academician, "a mixture of cells derived from a whole heart" may cause clinical risk of severe side effects on cardiac tissue (heart) of the recipient due to the existence of academician, if the mixture is used for transplantation in the heart without pre-treatment. Thus, for a more secure and reliable transplantation of cardiomyocytes to the recipient, it is preferable to increase the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture, i.e. to maximize the content of cardiomyocytes prior to transplantation. In addition, for example, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

To accurately increase the content of cardiomyocytes in this way after stage (1) and before stage (2) the method according to the present invention may further include a stage of cultivation labeled cells in the absence of a mitochondrial indicator. This stage provides the increase of the relative intracellular fluorescence intensity of cardiomyocytes relatively low level of fluorescence intensity of other types of cells, and more precisely leads to an increase in the number of cardiomyocytes.

As described above, a mitochondrial indicator, the use of which has been created in this embodiment of the present invention, can be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, the present invention M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In addition, in the third embodiment of the present invention the present invention relates to a method for producing cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes, where the method includes the following stages:

(1) the stage of differentiation and stimulation of obtaining cardiomyocytes from cells with the capacity to differentiate into cardiomyocytes with a receipt containing the cardiomyocytes cell mixture;

(2) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(3) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

In the context of this case for containing the cardiomyocytes mixture of cells may be a mixture of cells derived from a whole heart or a mixture of cells derived from cells with the capacity to differentiate into a cardiomyocyte. As used in the camping here the term "mixture of cells derived from a whole heart" means a mixture of cardiomyocytes and academician, "a mixture of cells derived from a whole heart" may cause clinical risk of severe side effects on the cardiac tissue of the recipient (heart) due to the existence of academician, if the mixture is used for transplantation in the heart without any pre-processing. Thus, for a more secure and reliable transplantation of cardiomyocytes to the recipient, it is preferable to increase the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture, i.e. to maximize the content of cardiomyocytes prior to transplantation. In addition, for example, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

For error-free receipt of cardiomyocytes in this way after stage (1) and before stage (2) the method according to the present invention may further include a stage of cultivation labeled cells in the absence of a mitochondrial indicator.

As described above, the mitochondrial indicator used in this embodiment can also be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, 302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In this area as a way of differentiation and stimulation of the formation of cardiomyocytes revealed that the formation of cardiomyocytes may occur during differentiation and stimulation of pluripotent stem cells that are less differentiated and have various possibilities of differentiation. The term "pluripotent stem cell" is defined as a cell with the ability to proliferate indefinitely or over a long period of time in culture conditionsin vitrobe maintained in the undifferentiated state, with a normal karyotype (chromosome) and having the ability to differentiate into any cell Rostock any of the three sheets (ectoderm, mesoderm and endoderm) in appropriate circumstances. Currently as pluripotent stem cells in this area is well known three types of cells, i.e. embryonic stem cells (ES cells), which secrete of the early embryo, embryonic germ cells (EG cells), which secrete from the primary germ cells in embryonic stages and adult pluripotent stem cells (also nasyvayevsky multipotential cells precursors (MAPC)), which are extracted from adult bone marrow.

For example, the method of differentiation and stimulation of the formation of cardiomyocytes from embryonic stem cells (for example, pluripotent stem cells may be selected from the group consisting of a movable cultural embryonic units, like Taurus, drip culture, culture with feeder cells culture with rotation, culture in soft agar and culture microneedle.

For example, in the case of the mobile culture of embryonic aggregates, similar to the Taurus, it is known that the Autonomous pulsating cardiomyocyte can be obtained by differentiation and stimulation of ES cells, where the method includes the following stages: the stage of suspension of embryonic stem cells in culture medium at concentrations of a few hundred cells/ml, where each cell ES is in the form of individual cells (i.e. in the state in which all cells are distributed in the aqueous phase without intercellular adhesion using an enzymatic treatment), the stage of culturing cells in suspension culture in the absence of a factor that inhibits differentiation (such as factor inhibiting leukemic cells: LIF), the stage of structure formation, such as the early embryo, known as embryonic body (EB), which is formed by the adhesion and aggregation of ES cells between the Wallpaper, and the stage of cultivation EB in terms of suspension culture or attached culture.

In the case of drip culture known that Autonomous pulsating cardiomyocyte can be obtained by differentiation and stimulation of ES cells, where the method includes the following stages: stage for receiving droplets consisting of 20 μl of culture medium, containing a few hundred cells on the lid of the Cup for cultivation, the phase space of the cover Cup for cultivation with drop Cup for cultivation, the stage of formation of cell mass in the lower part (i.e. at the top) drops, and the stage of differentiation and stimulation of the formation of Autonomous pulsating cardiomyocytes from the cell mass.

In the case of culture with feeder cells it is known that the Autonomous pulsating cardiomyocyte can be obtained by differentiation and stimulation of ES cells, where the method includes the following stages: stage for receiving a feeder layer of cells with properties similar to mesenchymal cells, preferably cells with properties similar to stromal cells in the bone marrow (such as ST2 cells, OP9 cells, cells PA6), using a method such as culture, high density, treatment with mitomycin C or radiation exposure, and the stage of culturing ES cells, ahogadas is in the form of individual cells on feeder layer.

In addition, in the fourth embodiment, the present invention relates to a method of estimating the percentage of cardiomyocytes in containing the cardiomyocytes mixture of cells, where the method includes the following stages:

(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and

(2) the stage of determining the ratio of cardiomyocytes and academician based on the relative content of mitochondria in the cells and/or selection stage cardiomyocytes based on the relative transmembrane potential of mitochondria of cells.

In the context of this case for containing the cardiomyocytes mixture of cells may be a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes. As used here, the term "mixture of cells derived from a whole heart" means a mixture of cardiomyocytes and academician, "a mixture of cells derived from a whole heart" may cause clinical risk of severe side effects on the cardiac tissue of the recipient (heart) due to the existence of academician, if the mixture is used for transplantation in the heart without any pre-processing. Thus, for a more secure and reliable transplantation ka is cardiomyocytes recipient is preferable to increase the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture, i.e. to maximize the content of cardiomyocytes prior to transplantation. In addition, for example, the cell with the capacity to differentiate into a cardiomyocyte, can be selected from the group consisting of stem cells, cell precursor and egg.

Also in this embodiment, as described above, the mitochondrial indicator used in this embodiment of the present invention may be selected from the group consisting of A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 and T3168. In this embodiment, M7512, T3168, T668 or R302 are preferred as a mitochondrial indicator.

In the area associated with the transplantation of cardiomyocytes, there are several known methods for differentiation and stimulation of the formation of cardiomyocytes and it is expected that the number of ways will be developed in the future as a way of differentiation and stimulation of the formation of cardiomyocytes. However, as described above, when using a mixture of transplantation in the heart there is a clinical risk associated with serious adverse effects on cardiac tissue recipient (heart) due to the existence of academician. Thus, it is possible to pre-evaluate the safety of the drug cardiomyocytes, art is meant to be used for transplantation, by preliminary estimates of the percentage of cardiomyocytes in the product intended for transplantation into cardiac tissue in accordance with this method.

As methods of purification of cardiomyocytes in this area there are several known methods such as a method based on the interaction of antigen-antibody, such as a method of flow cytofluorometry, a method using magnetic beads, the method of panning and so on (Monoclonal Antibodies: principles and practice, Third Edition (Acad. Press, 1993); Antibody Engineering: A Practical Approach (IRL Press at Oxford University Press, 1996); the method of selection of cells with the phenotype of cardiomyocytes by pre-embedding artificial modifications in gene pluripotent stem cells as stem cells such as ES cells), to stabilize a drug or ability to ectopic expression of the protein; and a method of fractionation of cells by centrifugation in a density gradient using media, such as sucrose and percoll, which can be easily combined with other methods, despite the relatively low degree of purification (Circ Res. 2002 20;91:501-508), etc.

The EFFECTS of the INVENTION

In accordance with the present invention may be selected cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes. In addition, in compliance and with the present invention it is also possible to increase the number of cardiomyocytes in containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes and get cardiomyocytes without genetic changes cardiomyocytes. Moreover, it is also possible to estimate the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture obtained in various ways.

In accordance with these options implemented, it is possible to increase the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture and reduce the variety of possible side effects on the cardiac tissue of the recipient (heart) due to the existence of academician after transplantation.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the fluorescent image of the cell mixture derived from a whole heart extracted from the hearts of newborn rats, which were marked mitochondrial indicator M7512.

Figure 2 presents a fluorescent image of a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes derived from embryonic stem cells of mice that were marked mitochondrial indicator M7512.

Figure 3 shows the distribution of the cell population when tagging a mixture of cells derived from an extract of the whole hearts of newborn rats, mitochondrial indicator M7512, where the amount of fluorescence in all cells was determined separately.

Figure 4 presents the distribution of cell populations when tagging a mixture of cells derived from the cells, the region is giving the ability to differentiate into cardiomyocytes, derived from embryonic stem cells mouse mitochondrial indicator M7512, where the amount of fluorescence in all cells was determined separately.

Figure 5 presents a fluorescent image of a cell mixture derived from a whole heart (presented on figure 3), which were labelled with antibodies against marker of cardiomyocytes, antibodies against α-actinin of sarcomere.

Figure 6 presents a fluorescent image of a mixture of cells derived from the cells with the capacity to differentiate into a cardiomyocyte (presented on figure 4), which were labelled with antibodies against marker of cardiomyocytes, antibodies against α-actinin of sarcomere.

Figure 7 presents the fluorescent image of the cell population obtained using antibodies against marker of cardiomyocytes, antibodies against α-actinin of sarcomere, where the cell population is subjected distribution analysis and cell sorting with a separate determination of the magnitude of fluorescence for each cell after tagging mitochondrial indicator T3168 cell mixture derived from a whole heart, extracted from the hearts of newborn rats.

On Fig presents the result of analysis of the cell populations that were subjected to analysis of distribution and cell sorting with a separate determination of the magnitude of fluorescence for each cell of p is the following labeling of the mitochondrial indicator T668 cells, extracted from whole hearts of newborn rats, and fluorescent image of each cell population using antibodies against marker of cardiomyocytes, antibodies against α-actinin of sarcomere.

On Fig-1 presents the division of the cell population obtained from the hearts of newborn rats in 3 groups of cell populations on the basis of the fluorescence intensity of T668.

On Fig-2 shows that almost all cells of the cell population with the highest intensity of fluorescent signal T668 consist of cardiomyocytes and almost all cells of the cell population with an average intensity of fluorescence consist of academician.

On Fig-3 shows that the required cells can also be obtained using R302 as T668.

Figure 9 presents the analysis of the cell populations of embryonic cells of rats derived from a whole heart, using mitochondrial indicator T668 and immune staining of sorted cells by antibodies against actinin.

Figure 9-1 presents the separation of cell populations derived from the fetal heart rats for 13 days after fertilization, into 3 groups of cell populations on the basis of the fluorescence intensity of T668.

Figure 9-2 shows that almost all cells of the cell population with the highest intensity of fluorescence with the persecuted T668 consist of cardiomyocytes and almost all cells of the cell population with an average intensity of fluorescence consist of academician.

Figure 10 presents the tagging mitochondrial indicator T668 whole embryonic cells and purification of cardiomyocytes.

Figure 10-1 shows that the population of individual cells obtained from embryos of rats 9 days after fertilization, mainly detected in the allocation of cells by fluorescence intensity.

Figure 10-2 shows that more than 95% of cells labeled T668, identified as cardiomyocytes when the immune staining against marker of cardiomyocytes actinin.

Figure 11 presents data tagging and analysis of cell population of cells of rats, obtained from the whole heart, in the time period from 11 days after fertilization to 8 days after birth using a mitochondrial indicator T668.

On Fig presents data tagging mitochondrial indicator T668 population, consisting of many types of cells derived from embryonic stem cells of mice, and immune staining of sorted cell population against actinin.

OPTION of carrying out the INVENTION

(1) the Receipt containing the cardiomyocytes mixture of cells

Firstly, containing the cardiomyocytes mixture of cells obtained as a cell mixture derived from a whole heart or a mixture of cells derived from stem cells.

The mixture of cells derived from a whole heart, can b the th obtained by removing the ventricle from abortive embryo or newborn animal, grinding it using tweezers, remove intercellular adhesion using an enzyme, such as collagenase, for separating cells.

The mixture of cells derived from stem cells can be obtained by differentiation and stimulation of stem cells such as embryonic stem cells and somatic stem cell, obtaining cardiomyocytes using the drip method of culture (Bader A, et al., Differentiation, 2001 68: p.31-43). Preferably, when using the mixture obtained from stem cells, spent additional processing to increase the degree of differentiation/stimulation of the formation of cardiomyocytes (such as the addition of all-TRANS retinoic acid) in differentiating/stimulated cell mixture.

(2) Tagging of cells mitochondrial indicator

In accordance with the present invention as desired mitochondrial indicator uses a fluorescent substance, such as M7512, T3168, T668 or R302 (all supplied Molecular Probe). Cells mark M7512, T3168, T668 or R302 by incubation of cell mixture derived from a whole heart, or a mixture of cells derived from stem cells in the presence M7512, T3168, T668 or R302 in culture medium.

Additional culturing for several days after labeling cells mitochondrial indica is a PR, you can further increase the differences between the values of the signal from the label for cardiomyocytes and academician.

(3) Selection of cardiomyocytes

For measurement of the content of mitochondria in a cell mixture derived from a whole heart, or in a mixture of cells derived from stem cells, labeled M7512, T3168, T668 or R302 use of cell sorters and cell population containing a relatively higher number of mitochondria, isolated as cardiomyocytes. As the preferred cell sorting device for selecting cardiomyocytes using M7512, T3168, T668 or R302 used cell sorters with activated fluorescence (FACS (registered trademark); BD, Franklin Lakes, NJ USA).

(4) Acknowledgement of receipt of cardiomyocytes

Cells exhibiting relatively higher intensity of fluorescence, sorted as described above and carry out the cultivation. Then in order to confirm the effectiveness of the present invention calculated the content and the factor content of cardiomyocytes allocated presented method are compared with the values for cardiomyocytes, isolated by the methods of separation of cardiomyocytes from other types of cells, different from the presented method. In accordance with the present invention as another method of separation of cardiomyocytes from other types of cells can be used a method of determining cardiomyocytes using antibodies against specific the La cardiomyocytes marker (such as a heavy chain/light chain of myosin, α-Akinin of sarcomere, troponin I, ANP, GATA-4, Nkx2.5, MEF-2c). Mainly, in accordance with the present invention for tagging and selection of cardiomyocytes using antibodies against α-Akinina of sarcomere.

EXAMPLES

The following examples are provided to further illustrate the present invention. However, the following examples should not be viewed as limiting the technical scope of this invention, but only as an illustration.

EXAMPLE 1: Tagging mitochondrial indicator cell mixture derived from a whole heart, extracted from the hearts of newborn rats

This example was conducted to confirm the possibility of applying the method according to the present invention for determining cardiomyocytes in a cell mixture derived from a whole heart.

Newborn rats at 1-3 days after birth were killed by cervical vertebrae shift after anesthesia with ether, then extracted the heart of each rat. The ventricles isolated from the heart, and treated with 0.025% of (wt./about.) by collagenase (supplied Warthington Biomedical Corporation) in D-MEM (high glucose)containing no serum medium (supplied by Invitrogen). The splitting of the ventricles of the heart using collagenase cells were dispersible in an environment with a mixture of cells derived from a with the rdca.

Then the culture medium was replaced with medium D-MEM (high glucose) (Invitrogen) supplemented with 10% (final concentration) of fetal calf serum (JRH Bioscience). Thus, the cell culture is incubated in culture medium containing 100 nm (final concentration) of the mitochondrial indicator, M7512 (supplied by Molecular Probe) for 10 minutes at 37°C. After incubation, the cells were washed 4 times using the culture medium and was further cultured for 24 hours at 37°C.

Thus, supervised labeled cells using a fluorescent microscope. The results are presented in figure 1.

Figure 1 shows that while the fluorescence-based M7512 was clearly observed in cardiomyocytes and cardiomyocytes contained numerous mitochondria in the cell, decationized contained only a small number of mitochondria in the cell.

EXAMPLE 2: Labeling of mitochondrial indicator of a mixture of cells derived from embryonic stem cells mouse

This example was conducted to confirm the possibility of applying the method according to the present invention for determining cardiomyocytes in a mixture of cells derived from stem cells.

Nedifferencirovannaja embryonic stem cells from mice subjected Capel is further cultivation (Bader A, et al., Differentiation, 2001 68: p.31-43) for differentiation and stimulation of the formation of cardiomyocytes. Specifically, the known drip culture, which can be derived cardiomyocytes through differentiation and stimulation of ES cells is represented by a method, where the method includes the following stages: stage for receiving droplets consisting of 20 μl of culture medium on the lid of the Cup for cultivation, where the specified drop of a few hundred cells, the phase space of the cover Cup for cultivation with drop Cup for cultivation, the stage of formation of cell mass in the lower part of the drop, i.e. on top of the drops, and the stage of differentiation and receiving from the cellular mass of the Autonomous pulsating cardiomyocytes. In accordance with the present invention differentiation and stimulation of the formation of cardiomyocytes used medium α-MEM (SIGMA)containing 10% (final concentration) of fetal calf serum (EQUITECH-BIO).

Fourteen days after differentiation and stimulation confirmed the formation in the vessel for culturing the cell masses containing Autonomous pulsating cardiomyocytes, to which was added 10-8M all-TRANS retinoic acid to further increase the degree of differentiation/stimulation of cardiomyocytes.

At 21 days after differentiation/stimulation-cell mass that contains the offline pulsating cardiomyocytes were divided into individual cells with a mixture of cells. Thus obtained mixture of the cells were labeled M7512 in the conditions specified in example 1 and were cultured in attached culture within 5 days after the removal M7512. After further culturing for 5 days-cell mass that contains the offline pulsating cardiomyocytes obtained in the culturing conditions were observed using a fluorescent microscope. The results are presented in figure 2.

Figure 2 presents pictures of phase-contrast images (top panel) and fluorescence images (bottom panel) cell mass during one cycle surge (i.e. cycle of the phase relaxation phase 1 reduction - phase relaxation 2). These results show that all Autonomous pulsating cardiomyocytes were strictly outline M7512 compared to other cell types. Thus, fluorescence-based M7512 was clearly observed in the cellular mass composed of Autonomous pulsating cardiomyocytes; while fluorescence-based M7512 were not observed in the cells of the cell mass.

EXAMPLE 3: analysis of the distribution of cells labeled mitochondrial indicator, smesi cells, derived from a whole heart

This example was performed to determine the number of cardiomyocytes contained in a cell mixture derived from a whole heart in example 1.

The mixture of cells derived from a whole heart, received in accordance with the method described in example 1, and were marked M7512. Labeled a mixture of cells derived from whole hearts were subjected to analysis of the distribution of cells in the cell mixture derived from a whole heart using FACS (registered trademark) (BD, Franklin Lakes, NJ USA). The results are presented in figure 3.

Figure 3 FSC-A" horizontal axis displays the amount of cells. As shown in figure 3, the mixture of cells derived from a whole heart, can be divided into two groups of cells, one of which consists of cells with a relatively higher intensity of fluorescence (shown in figure 3 in the form of the field P5), and the other consists of cells with relatively lower fluorescence intensity (shown in figure 3 in the form of the field P6). In this example, the cell region P5 were sorted as cardiomyocytes and cells area P6 were sorted as academician, then all of them separately subjected to cultivation.

EXAMPLE 4: analysis of the distribution of cells in a mixture of cells derived from embryonic stem cells mouse using mitochondriale the indicator

This example was performed to determine the number of cardiomyocytes contained in the mixture of cells from embryonic stem cells mouse obtained in example 2.

The mixture of cells derived from embryonic stem cells mouse received in accordance with the method described in example 2, and were marked M7512. Labeled a mixture of cells derived from embryonic stem cells of the mouse were subjected to analysis of the distribution of cells using FACS (registered trademark) (BD, Franklin Lakes, NJ USA). The results are presented in figure 4.

Figure 4 FSC-A" horizontal axis displays the amount of cells. As shown in figure 4, the mixture of cells derived from embryonic stem cells of a mouse, can be divided into two groups of cells, one of which consists of cells with a relatively higher intensity of fluorescence (shown in figure 4 in the form of region P2), and the other consists of cells with relatively lower fluorescence intensity. In this example, the cell region P2 were sorted as cardiomyocytes and they were subjected to cultivation.

EXAMPLE 5: Tagging marker of cardiomyocytes cells selected from the cell mixture derived from a whole heart

This example was performed to determine the extent of the increase in the content of cardiomyocytes, present in the mixture of cells obtained is Oh from a heart method according to example 3.

Cell population region P5 and area P6, sorted by the method according to example 3 were cultured for 12 hours in medium D-MEM (SIGMA) supplemented with 10% (final concentration) of fetal calf serum (EQUITECH-BIO), followed by fixation with paraformaldehyde.

Then the fixed cells were labeled with a marker of cardiomyocytes, antibodies against α-actinin of sarcomere mice (supplied by SIGMA), which were visualized using antibodies goat against mouse conjugated with green fluorescent substance (supplied by Molecular Probes). The results are presented in figure 5. Figure 5 the results for a cell mixture derived from a whole heart, presented in the top panels, the results for cells isolated from the field P5 according to example 3, presented in the middle panels and the results for cells isolated from the field P6 according to example 3 are presented in the lower panels.

Before allocating cardiomyocytes the method according to the present invention cardiomyocytes recognized by antibodies against α-actinin of sarcomere, and other types of cells were mixed in the cell mixture, and the percentage of cardiomyocytes in the middle of all cells, was only 30%. On the other hand, more than 99% of the cells isolated from the field P5 was cardiomyocytes. In cells isolated from the field P6, was in small amounts, the creation of cardiomyocytes, the percentage of which was approximately equal to the percentage of cardiomyocytes before allocating presented method.

The results of this example clearly show that the method according to the present invention can be selected cardiomyocytes with a high degree of purification and their content can be increased in a cell mixture derived from a whole heart.

EXAMPLE 6: Tagging marker of cardiomyocytes cells taken from a mixture of cells derived from embryonic stem cells mouse

This example was performed to determine the extent of the increase in the content of cardiomyocytes, present in the mixture of cells derived from embryonic stem cells of the mouse by the method according to example 4.

Cell population region P2, and other areas, sorted by the method according to example 4 were cultured for 12 hours in medium D-MEM (SIGMA) supplemented with 10% (final concentration) of fetal calf serum (EQUITECH-BIO), followed by fixation with paraformaldehyde.

Then the fixed cells were labeled with a marker of cardiomyocytes, antibodies against α-actinin of sarcomere mice (supplied by SIGMA), which were visualized using antibodies goat against mouse conjugated with green fluorescent substance (supplied by Molecular Probes). The results are presented at the Phi is .6. Figure 6 the results for the mixture of cells derived from embryonic stem cells mouse presented in the top panels and the results for cells isolated from a region P2 in example 4, are presented in the lower panels.

Before allocating cardiomyocytes the method according to the present invention cardiomyocytes recognized by antibodies against α-actinin of sarcomere, and other types of cells were mixed in the cell mixture, and the percentage of cardiomyocytes in the middle of all cells, was only 10%. On the other hand, it was shown that more than 80% of the cells isolated from the area P2 (the relatively higher intensity of fluorescence when tagging M7512)are cardiomyocytes.

The results of this example clearly show that the method according to the present invention can be selected cardiomyocytes with a high degree of purification and their number can be increased in a mixture of cells derived from embryonic stem cells of the mouse.

EXAMPLE 7: Tagging mitochondrial indicator T3168

This example was carried out to confirm that when using the mitochondrial indicator T3168 for tagging cells to obtain data similar to the data obtained using M7512.

In this example, the mixture of cells derived from a whole heart, extracted from the hearts of newborn cu is si, which were marked mitochondrial indicator, obtained by the method described in example 1, except that as a mitochondrial indicator used T3168 (supplied by Molecular Probe).

Obtained and labeled thus a mixture of cells derived from a whole heart, to analyze the distribution of the cells were subjected to FACS as described in example 3, and cells with a relatively higher intensity of fluorescence was determined and allocated as cardiomyocytes. Underlined cells were cultured with subsequent fixation with paraformaldehyde as described in example 5, which were labelled with antibodies against α-actinin of sarcomere (supplied by SIGMA). The results are shown in Fig.7.

Before selecting cardiomyocytes the method according to the present invention cardiomyocytes recognized by antibodies against α-actinin of sarcomere, and other types of cells were mixed in the cell mixture, and the percentage of cardiomyocytes in the middle of all cells, was only 30%. On the other hand, it was shown that more than 95% of cells exhibiting relatively higher intensity of fluorescence are cardiomyocytes.

The results of this example clearly show that the method according to the present invention can be selected cardiomyocytes with a high degree of purification and their number can be the ü increased in the mixture of cells, derived from a whole heart, using T3168 as a mitochondrial indicator.

EXAMPLE 8: Tagging mitochondrial indicator T668 cells of newborn rats, obtained from the whole heart, and purification of cardiomyocytes

To obtain cell populations hearts of newborn rats were treated with 0.025% of (wt./about.) by collagenase (supplied by SIGMA) and trypsin (supplied by GIBCO). Cells dispersed in the culture medium was subjected to 1 μm (final concentration) of the mitochondrial indicator T668 (supplied by Molecular Probe) for 15 minutes at 37°C, washed 3 times and immediately analyzed using FACS. Then the cells were divided into three groups in accordance with the fluorescence intensity on the basis of T668 (Fig-1). Cell population with high fluorescence, showing a higher intensity of fluorescence, and cell population with an average fluorescence were sorted separately.

Then the immune staining of cell cultures using antibodies against actinin identified cardiomyocytes (Fig-2). Further, almost all cells in the cell population with the highest fluorescence signal T668 consisted of cardiomyocytes, and almost all cells in the cell population with an average intensity of fluorescence consisted of academician. The decree of the config analysis was carried out in respect of R302 (supplied by Molecular Probe) and obtained similar results (Fig-3), as for T668.

EXAMPLE 9: the Tagging of the cells of newborn rats, obtained from a heart mitochondrial indicators T668 and M7514, and comparative analysis of the transmembrane potential of mitochondria

Cell populations were isolated by using the materials and method described in example 8. Underlined cardiomyocytes were marked mitochondrial indicator M7514 (supplied by Molecular Probe), independent of the transmembrane potential, which is specific marks mitochondria, and mitochondrial indicator T668-dependent transmembrane potential, which is also specific marks mitochondria. Cells were analyzed using FACS immediately after tagging. As the wavelength of fluorescence T668 is different from the wavelength of the fluorescence M7514, it is possible to separately determine the fluorescence T668 and fluorescence M7514. In this example, identified three groups of cell populations on the basis of fluorescence T668 as an indicator. As described for analysis according to example 8, it is known that the cell population with high fluorescence, showing the highest fluorescence intensity, is a population of cardiomyocytes, and that the population with the average fluorescence represents the population of academician.

Then each cell population was divided based on the relative intensity of fluorescent and, obtained using T668 and fluorescence intensity obtained for M7514. In cell population identified as cardiomyocytes based on the signal T668, the number of cells assigned to a group with a value of more than 150%, accounted for 90% of the total number of cells using limiting values of the ratio of the fluorescence intensity of T668 and fluorescence intensity M7514 equal to 150%; while in the cell population identified as decationized signal-based T668, the number of cells assigned to a group with a value of more than 150%, accounted for 13% of the total number of cells using limiting values of the ratio of the fluorescence intensity of T668 and fluorescence intensity M7514 equal to 150%. These results indicate that cardiomyocytes have not only a higher content of mitochondria, but also a higher transmembrane potential of mitochondria compared to academicaly.

EXAMPLE 10: the Tagging of mitochondrial indicator T668 embryonic cells of the rat, derived from a whole heart, and purification of cardiomyocytes

Heart of the embryo rats for 13 days after fertilization were treated with collagenase and trypsin to obtain cell populations. Cells dispersed in the culture medium was subjected to 1 μm (final concentrat the I) mitochondrial indicator T668 for 15 minutes at 37°C, washed 3 times and once was subjected to FACS analysis. The cells were divided into 3 groups of cell populations on the basis of the fluorescence intensity of T668 (Fig.9-1). In this example, the cell population with high fluorescence, showing the highest fluorescence intensity and the cell population with an average fluorescence were sorted and cultured.

Then the immune staining of cell cultures using antibodies against actinin identified cardiomyocytes. Further, almost all cells in the cell population with the highest fluorescence signal T668 consisted of cardiomyocytes and almost all cells in the cell population with an average intensity of fluorescence consisted of academician (Fig.9-2).

EXAMPLE 11: the Tagging of mitochondrial indicators T668 and M7512 whole embryonic cells and purification of cardiomyocytes

The embryo rats 9 days after fertilization were treated with collagenase and trypsin to obtain a cell population. Cells dispersed in the culture medium was subjected to 1 μm (final concentration) of the mitochondrial indicator T668 for 15 minutes at 37°C, washed 3 times and once was subjected to FACS analysis. As the cell population on the basis of fluorescence intensity were mainly observed one individual is gunning population. On the other hand, in areas of high fluorescence intensity, which is supposed to detect cardiomyocytes based on the analysis of a sample of the whole heart at a later stage of development, it was impossible to identify a specific cell population (figure 10-1). However, since there were a small number of cells exhibiting a higher fluorescence intensity than in the main cell population, these cells were identified as the cell population with high fluorescence.

The granulosa cells and immunologically stained with antibodies against marker of cardiomyocytes actinin. It was found that approximately more than 95% of cells allocated using T668, represented the cardiomyocytes (figure 10-2). As in the embryo at 9 days after fertilization (early embryo) cardiomyocytes are very immature in this area believe that quantitative changes in the mitochondria is not absolute. These data show that, despite the early stage, it is possible to effectively allocate cardiomyocytes using the transmembrane potential of mitochondria as an indicator.

EXAMPLE 12: Staining of cells in rats, obtained from the whole heart, in the period from 11 days after fertilization to 8 days after birth, using a mitochondrial indicator T668 and FACS analysis

To obtain a cell population heart rats, obtained in the period from 11 days after fertilization to 8 days after birth, were treated with collagenase and trypsin. Cells dispersed in the culture medium was subjected to 1 μm (final concentration) of the mitochondrial indicator T668 for 15 minutes at 37°C, washed 3 times and immediately analyzed using FACS. Then cell populations were isolated by fluorescence intensity T668. At higher stages of development, the population of cardiomyocytes with high fluorescence, showing the highest fluorescence intensity, were numerically increased, and cell populations clearly differ among themselves (11). Thus, it is shown that it is possible to make an assumption about the stage of maturation of cardiomyocytes method presented in the present description.

EXAMPLE 13: Tagging mitochondrial indicator T668 population consisting of various types of cells, and purification of cardiomyocytes

Spent the differentiation of ES cells in the cell population containing the cardiomyocytes using the method specified in example 2. To obtain single cell this cell mass, consisting of various types of cells, were treated with collagenase and trypsin. Cells dispersed in the culture medium was subjected to 1 MK is (final concentration) of the mitochondrial indicator T668 for 15 minutes at 37°C, washed 3 times and immediately analyzed using FACS. As the cell population on the basis of fluorescence intensity were mainly observed single cell population. On the other hand, in areas of high fluorescence intensity, which is supposed to detect cardiomyocytes based on the analysis of a sample of the whole heart at a later stage of development, it was impossible to identify a specific cell population (Fig). However, since there were a small number of cells exhibiting a higher fluorescence intensity than in the main cell population, these cells were identified as the cell population with high fluorescence.

Cells were cultured in a large scale and immunologically stained with antibodies against marker of cardiomyocytes actinin. It was revealed that more than 98% of the cells represented the cardiomyocytes. On the other hand, the main cell population also were isolated and cultured with subsequent immune staining with antibodies against marker of cardiomyocytes actinin. Almost all the cells consisted of academician.

EXAMPLE 14: Tagging mitochondrial indicator S7563 cells of newborn rats, obtained from the whole heart, and purification of cardiomyocytes

Heart of newborn rats was treated to what lagente and trypsin to obtain a cell population. Cells dispersed in the culture medium was subjected to 1 μm (final concentration) of the mitochondrial indicator S7563 for 15 minutes at 37°C, washed 3 times and immediately analyzed using FACS. Then the cells were divided into 3 groups of cell populations on the basis of fluorescence intensity S7563. In this example, the cell population with high fluorescence, showing the highest fluorescence intensity, were sorted and cultured.

Then the cardiomyocytes were identified by immune staining of cell cultures using antibodies against actinin. Further, almost all cells in the cell population with the highest fluorescence signal S7563 consisted of cardiomyocytes and almost all cells in the cell population with an average intensity of fluorescence consisted of academician.

INDUSTRIAL APPLICABILITY

In accordance with the present invention may be selected cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes. In addition, in accordance with the present invention, it is possible to increase the content of cardiomyocytes in containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes and get cardiomyocytes without genetic changes of cardiomyocytes. More tor the, you can also estimate the percentage of cardiomyocytes in containing the cardiomyocytes cell mixture obtained in various ways.

In accordance with these options implemented, it is possible to increase the percentage of cardiomyocytes in containing the cardiomyocytes mixture of cells and reduce the various possible side effects on the cardiac tissue of the recipient (heart) due to the existence of academician after transplantation.

1. The method of selection of cardiomyocytes from containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes based on the relative content of mitochondria in the cells, the relative transmembrane potential of mitochondria of cells, or as the relative content of mitochondria in cells, and the relative transmembrane potential of mitochondria of cells, where the method involves the step of labeling of the mitochondrial indicator containing the cardiomyocytes mixture of cells and the stage of measuring the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells and cardiomyocytes isolated in accordance with the following: cell population with a relatively high content of mitochondria; cell population containing mitochondria with relatively high transmembrane potenziale is; or cell population comprising cells having a relatively high content of mitochondria and relatively high transmembrane potential.

2. The method according to claim 1, where the method additionally includes the stage of cultivation labeled cells in the absence of a mitochondrial indicator after stage labeling of mitochondrial indicator containing the cardiomyocytes mixture of cells at the stage of measuring the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

3. The method according to any one of claims 1 and 2 where the specified containing the cardiomyocytes mixture of cells is a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes.

4. The method according to claim 3, where the cell with the capacity to differentiate into a cardiomyocyte selected from the group consisting of stem cells, cell precursor and egg.

5. The method according to any one of claims 1, 2 or 4, where the specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T, T, T and T.

6. The method according to claim 3, where the specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D38, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T,T,Tit.

7. The way to increase the content of cardiomyocytes in containing the cardiomyocytes mixture of cells without genetic changes of cardiomyocytes, comprising the following stages:
(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and
(2) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells
where the specified method further includes a step of cultivation of labeled cells in the absence of a mitochondrial indicator after stage (1) and before stage (2).

8. The method according to claim 7, where the specified containing the cardiomyocytes mixture of cells is a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes.

9. The method of claim 8, where the specified cell with the capacity to differentiate into a cardiomyocyte selected from the group consisting of stem cells, cell precursor and egg.

10. The method according to any of claim 7 or 9, where the specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M,R302, R634, R648, R14060, R22420, T, T, T and T.

11. The method of claim 8, where the specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T, T, T and T.

12. A method of producing cardiomyocytes without genetic changes of cardiomyocytes, comprising the following stages:
(1) the stage of differentiation and stimulation of the formation of cardiomyocytes from the cell with the capacity to differentiate into cardiomyocytes with a receipt containing the cardiomyocytes mixture of cells;
(2) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and
(3) selection stage cardiomyocytes based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

13. The method according to item 12, where the method additionally includes the stage of cultivation labeled cells in the absence of a mitochondrial indicator after stage (2), prior to stage (3).

14. The method according to item 12 or 13, where the specified cell with the capacity to differentiate into a cardiomyocyte selected from the group consisting of stem cells, cell precursor and egg.

15. The method according to item 12 or 13, where the specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426,D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T, T, T and T.

16. The method according to 14, where specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T, T, T and T.

17. Method of assessment content in cardiomyocytes containing the cardiomyocytes cell mixture, comprising the following stages:
(1) the stage of labeling containing the cardiomyocytes cell mixture mitochondrial indicator; and
(2) the stage of determining the ratio of cardiomyocytes and academician based on the relative content of mitochondria in the cells and/or the relative transmembrane potential of mitochondria of cells.

18. The method according to 17, where specified containing the cardiomyocytes mixture of cells is a mixture of cells derived from a whole heart, or a mixture of cells derived from cells with the capacity to differentiate into cardiomyocytes.

19. The method according to p where the specified cell with the capacity to differentiate into a cardiomyocyte selected from the group consisting of stem cells, cell precursor and egg.

20. The method according to any of PP-19, where specified mitochondrial indicator selected from the group consisting of A, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M, M, M, M, M, M, M, M, M, M, R302, R634, R648, R14060, R22420, T, T, T and T.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method of detection of electric myocardium instability risk with unstable stenocardia is based on defining the following parameters for patients with unstable stenocardia: erythrocyte sedimentation rate; stab neutrophil content; aspartate aminotransferase and alanine aminotransferase activity; general cholesterol level; carbamide, creatin, β-lipoproteid content; glucose and fibrinogen concentration. For each parametre, parametre element value of electric myocardium instability risk is calculated. Risk indicator for electric myocardium instability is calculated as arithmetic mean of the elements, and if the value exceeds 0.4, then risk of electric myocardium instability development is diagnosed for unstable stenocardia.

EFFECT: possible detection of electric myocardium instability risk.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves defining cytochrome C and antibody titre to herpes virus by enzyme immunoassay method in placental homogenate. At cytochrome C concentration of 13.9±0.06 pg/ml and antibody titre to herpes virus of 1:6400, it is diagnosed that 1.5±0.002% of placental stem symplast nuclei are in apoptosis state; at cytochrome C concentration of 18.8±0.08 pg/ml and antibody titre to herpes virus of 1:12800 it is diagnosed that 3.0±0.03% of placental stem symplast nuclei are in apoptosis state.

EFFECT: high sensitivity of method even for nuclei at the very beginning of scheduled death, possible forecast of apoptosis development in symplast nuclei of placental stems.

FIELD: medicine.

SUBSTANCE: invention can be used for tumor visualisation using hyperpolarisable 13C-pyruvate as magnetic resonance imaging agent, which can distinguish healthy tissue from tumor tissue. The following shall be performed: a) acquisition of direct 13C-MP images of 13C-pyruvate and its 13C-containing metabolites of alanine and lactate in the patient previously injected with composition containing hyperpolarisable 13C-pyruvate, b) correcting lactate signal with respect to quantity of pyruvate and/or alanine with acquisition of lactate relative to pyruvate and/or lactate relative to alanine image whereby tumor tissue is indicated by the highest signal and/or high weighted lactate signal relative to pyruvate signal and/or lactate signal relative to alanine signal in specified13C-images.

EFFECT: improved imaging accuracy when distinguishing healthy tissue from tumor tissue.

8 cl, 2 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to gastroenterology, and can be applied in prediction of gastropathies induced by non-steroid anti-inflammatory preparations. In complex clinic-functional criteria are determined: morbid load, age over 50 years, presence of accompanying diseases, reduction of liver ecogenicity in USE; blood fibrinogen lower than 4.0 g/l, increase of activated partial thromboplastin time (APTT) relative to norm, reduction of fibrinolysis time lower than normal, increase of creatinine in blood higher than 85 mcmole/l, level of antibodies (IgG) to Helicobacter pylori by data of immune-enzyme analysis lower than 2.0, blood leptin higher than 50 mg/ml, activity of blood antithrombin III is lower than normal, level of soluble fibrin-monomer complexes (SFMC) is higher than normal, level of aspartataminotransferase (AST) is higher than normal, C-reactive protein is higher than normal. Each criterion is given "+1" if it is present, and "0" if it is absent, and if sum of criteria is from "0 to 2", low degree of gastropathy development is determined, "from 3 to 5" - medium risk degree, and if value of criteria sum is "6 and higher" - high degree of gastropathy development.

EFFECT: defining 3 groups of risk of NAIP-gastropathies allows to improve approach to management and treatment of patients, taking NAIP, by means of rational use of selective COG - 2 inhibitors, constant therapy with proton pump inhibitors, carrying out FGDS of all patients possessing risk factors.

3 tbl, 5 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to veterinary science. An express diagnostic aid for urolithiasis in cats is indicator granules containing a base and a mixed indicator with said dry mixed indicator and base taken in the ratio 0.01-0.04. The base is fractionated Circulite coated with a mixed indicator fluid containing as follows, wt %: distilled water - 50-89.9%; a water-soluble polymer 10-49.9%; a water-soluble pH indicator - 0.01-0.1 %. The water-soluble polymer is either polyvinylpyrrolidone, or polyethylene glycol, or polymethacrylamide, or polyvinyl alcohol. The water-soluble pH indicator is phenol red and/or cresol red, and/or chinoline blue, and/or rosolic acid, and/or corallin algae, and/or nitrasin yellow, and/or curcumine. The method consists as follows: The mixed indicator fluid is solubilised. The prepared mixed indicator coats the base of Circulite granules fractionated by nozzle spraying at discharge 100-200 ml/min and at sprayed solution temperature 20-40°C. The granules are dried up to humidity 1-5% at temperature +40 to +250°C during 5-30 minutes to ensure the ratio of the dry mixed indicator to the base within 0.01-0.04.

EFFECT: enabled express diagnostics of urolithiasis.

4 cl, 4 ex

FIELD: medicine.

SUBSTANCE: method concerns medicine, more specifically venereology, and can be used in therapy of syphilis. The efficacy of sodium benzylpenicilline preparations in syphilis therapy has been estimated by evaluating antibiotic blood concentration. The inefficacy of therapy provided concentration less than 0.018 mkg/ml, but also more than 0.4 mkg/ml is deduced from experiments.

EFFECT: higher information value of the method.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to cardiology, and can be used in predicting the development of metabolic syndrome in male arterial hypertension. The patient with arterial hypertension is examined for biomedical measurements including cell membrane stability and predicted level of metabolic syndrome by following formula:

where K is a prognostic coefficient, CMS is cell membrane stability (sec), LDLPCL is low-density lipoproteid cholesterol (mmol/l), CL is total cholesterol (mmol/l), HDLPCL is high-density lipoproteid cholesterol (mmol/l). The K≤1.2 allows predicting high probability of metabolic syndrome development in male arterial hypertension, the K≥2.5 shows low probability of metabolic syndrome in male arterial hypertension, and in case of 1.2<K<2.5, average probability of male arterial hypertension is predicted.

EFFECT: development of exact prediction procedure for metabolic syndrome development.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biology, namely to sterility diagnostics. There is offered method for evaluating the reproductive ovarian potency. Ovarian tissue is fixed in Bouin's fixative solution; histological sections of thickness 7-8 mcm are prepared and stained in hematoxylin and eosin. A portion of germ cells is evaluated for each developmental stage from total gamete count: for oocytes in %% of total oocyte count; for follicles and derivatives - count at each stage per 1 microscopic field. The control indices of various oogenesis and folliculogenesis stages are determined in healthy representatives of different mammals. The reproductive ovarian potency and potential pathology are estimated after considering the results of comparison to the control indices.

EFFECT: possibility to diagnose impaired oogenesis and folliculogenesis infringements in gynaecology, veterinary science and livestock sector.

4 dwg, 8 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to nephrology and can be used for sclerosed glomerule count in haemorrhagic nephrosonephritis. The ratio of total natural logarithms of following indices is calculated including: ultrasound-aided averaged renal volume, cm3 (Vsr), as total renal volume divided by 2 (Vs+Vd)/2; hyaline casts count per mm2 of a cytologic section (D); number of burned-out and necrotic tubule epithelium per 1 mm2 of a cytologic section (D). Duration of acute renal failure, days (T), to total natural logarithms of glomerular filtration rate, ml/min (GFR) and averaged linear blood velocity in both basilar renal arteries (Vavesr) as (Vaves+Vaved/2), cm/sec. Then the glomerular sclerosis index (GSI) is calculated by formula: If the GSI exceeds 1.5, glomerular sclerosis is considered to develop, while the GSI less than 1.5 shows the absence of glomerular sclerosis in haemorrhagic nephrosonephritis.

EFFECT: method provides ease of examination with reduced number of nephrobiopsies in the present disease.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to laboratory diagnostics and can be used for evaluation of phagocytic activity of neutrophils in human peripheral blood. Phagocytosis objects are recombinant luminescent bacteria, namely microorganisms Escherichia coli with cloned luxCDABE genes Photobacterium leiognathii, pre-opsonised during 10 min with normal human immunoglobulin in end concentration 6-10 mg/ml. Luminole is additionally introduced to the sample to end concentration 10-3 M. Intensity of luminole-dependent phagocytic chemoluminescence and bioluminescence of bacterial targets are counted separately by evaluating luminescence in two various spectral bands: <430 nm for evaluation of chemoluminescence and >540 nm for evaluation of bioluminescence.

EFFECT: possibility for complex evaluation of activation level of oxygen-dependent bactericidal phagocytic systems, and improved test accuracy.

1 ex, 1 tbl

FIELD: chemistry, biochemistry.

SUBSTANCE: method can be used in microbiology, food industry for estimation of viability of unicellular organisms (yeasts, etc.), which demonstrate difference of dielectric properties. Per cent content in mixture of live and dead unicellular microorganisms is determined by deviation of measured value of dielectric permeability from dielectric permeability of mixtures, consisting only of live and only of dead unicellular microorganisms, or by experimental dependence of second derivative of dielectric permeability on humidity.

EFFECT: elaboration of distant methods of assessment in continuous flow, in elaboration of industrial methods of control over live microorganism production.

2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medical microbiology. The method of chronic urogenital gonococcal infection course forecast involves seeding of accompanying fungi of Candida genus in case of gonococcus detection, and persistence factors of accompanying microorganisms is evaluated. If titration of fungi of Candida genus gives not less than 102 colony-forming cells per millilitre and antilysozyme activity evolves simultaneously in the quantity not less than 1.3 mcg/ml per optical density unit, and anticomplementary activity not less than 1.5·106 antilytic complement units, then chronic character of urogenital infection is confirmed diagnosed.

EFFECT: increased accuracy of chronic urogenital gonococcal infection course forecast.

2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves carrying out bacteriological study of esophageal mucous membrane biopsy samples. No microorganism growth or predominant Streptococcus spp., Peptostreptococcus spp., Staphylococcus spp. in monoculture or culture association in the amount of equal to or greater than 103-104 CFU/g (colony formation units), and no Escherichia coli, Bacteroides spp., Enterococcus faecalis, Enterococcus faecium, Candida spp. in monoculture or culture association in the amount of equal to or greater than 102-107 CFU/g, and optionally increased total microorganisms quantity to 104-107 CFU/g being observed, alkaline ingredient availability in refluxate in gastroesophageal reflux cases is declared to take place.

EFFECT: enabled alkaline ingredient availability and microbiocenosis disorders intensity evaluation.

FIELD: medicine.

SUBSTANCE: method involves determining duodenal juice acidity, duodenum bulbary and postbulbary department insemination degree with H.pylori. Ulcer edge and pyloric canal area bioptates are subjected to immunological and histological examination. Duodenal juice acidity being equal to or higher than 6.5 mmole/h and duodenal mucous membrane insemination degree being equal to or higher than 100 bacteria in vision field, IgG antibodies to H.pylori diluted in 1:160 proportion and higher, gastric metaplasia being detected in pyloric canal bioptates of ulcer edges among hypertrophied smooth muscle cells of separate groups of atrophied and deformed smooth muscle cells divided by layers of loose connecting tissue having blood vessels, fibroblasts, lymphocytes and macrophages, and anisochromia being detected when staining hypertrophied smooth muscle cells, pylorostenosis development is to be predicted.

EFFECT: high accuracy in predicting pylorostenosis development clinical course.

FIELD: medicine.

SUBSTANCE: method involves separating pure microorganism cultures from nasal mucous membrane and/or rhinopharynx microflora and identifying them. Anti-lysozyme activity is determined in pure culture and microbial insemination share in the general microbial insemination index is calculated for biotope under study. The first value being equal to or greater than 3 mcg/ml and the second one greater than 45%, rhinotubal microorganism migration into middle ear tympanic cavity is to be predicted.

EFFECT: high accuracy in predicting clinical course of inflammation in middle ear.

2 tbl

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves sowing out samples of mixed cultures in liquid selective media, determination of cells number accumulating in media during microorganisms growth by bioluminescent method and mathematical treatment of kinetic data of the growth of individual bacterial cultures, determination of the parent concentration of microorganisms relating to different taxonomic groups. Invention allows carrying out the simultaneous identification of bacterial cells relating to different taxonomic groups and presenting in mixed cultures simultaneously, and to enhance precision in determination of cells number in the broad concentration range and to reduce the total analysis time significantly. In industry mixed cultures are used widely in different branches of food industry (dairy, meat, brewing and others) and in other biotechnological processes (biological treatment of sewage waters, bioremediation of soils, producing methane from waste in different manufactures and others). Invention can be used for differentiated determination of microorganisms number in mixed cultures that are widely distributed in nature: in air, soil, ponds, as components of natural microflora in higher organisms and among contaminants causing injury of different objects.

EFFECT: improved method for determination.

1 tbl, 3 dwg, 5 ex

FIELD: microbiology, optics.

SUBSTANCE: invention relates to investigations of materials by assay of their physical or chemical properties using optical devices and to systems wherein material is excited by optical agents resulting to it luminescence. Invention proposes a test carrier as a centrifugal tube. Carrier is separated for upper and bottom cavities by partition. Volume of lower cavity is 0.1 of tube volume. A hole is made in partition near a wall. The constructive decision of partition provides efflux of sample from lower cavity with minimal overcoming the combined forces of wetting and surface tension. Also, invention proposes methods/variants for rapid measurement of absolute concentration of microorganisms in biosubstrate by their photoluminescence. Methods involve using fluorescent or phosphorescent measuring device and above said test carrier. Methods provides increasing rate and precision of assay, to use serial measuring devices and to carry out measurement of the concentration of particles in substrate with another specific gravity value as compared with that of liquid in substrate. Invention can be used in food and biotechnological industry for determination of absolute concentration of microorganisms in different substrates.

EFFECT: improved method for assay, valuable properties of carrier.

2 tbl, 1 dwg

The invention relates to food industry, in particular to the baking industry, and can be used to determine the number of fungi on the surface of bakery products

The invention relates to the technical Microbiology and can be used in the oil industry for research oilfield environments on the content of sulfate reducing bacteria (SRBs) and the selection of biocide to inhibit the growth of sulfate reducing bacteria

FIELD: medicine.

SUBSTANCE: connective or supporting tissue cells fit for transplantation are cultivated in bioreactor (1) which includes main case closed by sterile sealing cap (21) and forms at least one reactor space where transplant surface (11) and mini actuator (14) are placed. Bioreactor (1) is equipped with at least two coupling connections for hoses (19) of nutrient medium and gas feed and discharge.

EFFECT: obtaining cells with mechanical endurance to self-tissue transplantation for application as substitute tissue in treatment of connective and supporting tissues, direct injury of joints, rheumatism and degenerative diseases of joints.

22 cl, 27 dwg, 11 ex

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