Method of single-step telomere length and population division quantity measurement of proliferative cells in vitro
SUBSTANCE: method involves telomere length measurement by flow-FISH method in cells differentiated by a number of population divisions with a vital stain without preliminary recovery of proliferative cells with using Bis(sulfosuccinimidyl)suberat (BS3) amino group cross-linker.
EFFECT: invention provides the single-step telomere length measurement in cells with a common division quantity.
3 dwg, 1 ex
The invention relates to the study of biological material, in particular to the determination of telomere length.
Telomeric regions of DNA (telomeres) are the ends of chromosomes that do not carry the semantic load, which consist of repeated hexaprotodon TTAGGG having a tandem arrangement and identical for all vertebrates [1, 8]. During cell division in the end naturalisatie DNA newly synthesized sister chromatid becomes shorter, and this happens due to the loss of telomeric repeats . Telomere shortening is a limitation to continuous cell division, therefore, telomere length determines the replicative potential of cells, and is an indirect marker of its replicative history. Short telomeres are a sign of "senescent" cells.
Telomere length is one of the main dynamic features of cell activity, and the study of such changes is an important fundamental and clinical significance. The change in telomere length with age and the possibility of its regulation are of great importance in studying the biology of aging and gerontology [2, 6]. Widely used measurement of telomere length in the diagnosis, evaluation of treatment efficacy and prognosis of cancer and conditions associated with disorders of the immune system [4, 13, 5]. The study of telomeres at the cellular level, changes associated with cell division, are of particular importance for understanding the biology of aging, as it affects the processes that determine the lifespan of the cell.
Measurement of telomere length has become a routine method by developing methods Flow-FISH-in situ hybridization analysis on a flow cytometer. This method allows to determine telomere length in single cells of the target population . Measurement of telomere in proliferating cells is possible in several ways. The most simple is the selection of proliferating cells using DNA dyes, when cells in phase S-G2-M have a greater number of DNA that allows them to differentiate at a higher level of fluorescence channel luminosity DNA dye . This technique included in method of Flow-FISH to measure telomere, so there is no need for separate sorting of proliferating cells. However, this method allows to determine the length of telomeres only in cells that were shared at the time of the study, and does not take into account cells that have shared earlier.
Another method is incubation of living cells in a solution containing bromodeoxyuridine (BrdU), which is incorporated into the newly synthesized DNA chain. Then add anti-BrdU monoclonal antibodies labeled with fluorochromes and analyze the on flow cytometer. This allows you to precisely select all shared cells, because they contain built-labeled nucleotide regardless of the time of the study.
Another method taken by the authors for the prototype that we can identify proliferating cells using vital fluorescent dye. The principle of the method lies in staining cells with the vital dye, which is strongly associated with blood cells, and after division is distributed between daughter cells . Nepodelishsya cells have a maximum luminance channel fluorescence of the vital dye, and cells, making at least one division will have the level of fluorescence is much lower. To determine telomere length in proliferating cells, it is necessary first to distinguish from the General population, i.e. sort, for example, at the cellular case, because the fluorochromes can not withstand heat up to 80°C, necessary for carrying out the method of in situ hybridization [3, 5, 14]. This method, like the previous one, a very expensive and time-consuming, requiring considerable time and, apart from the special cell sorting device, it is necessary to prepare a large number of cells.
Staining cells with the vital dye also allows you to determine the exact number of committed cell divisions in culture. After division of the mother cell dye R is Vomero distributed between daughter cells, who have the level of fluorescence in two times lower than that repeats itself with each new division. The number of peaks of fluorescence of the vital dye, you can determine the number of cell divisions .
The authors conducted a series of experiments in which it was shown that the vital dye CFSE not withstand heat up to 80°C in formamide, and after in situ hybridization is impossible to determine the number of divisions by the number of separate peaks on the channel fluorescence of CFSE. There are ways to save fluorochrome on the cell when heated up to 80°C, one of which is the use of special chemicals BS3 (Bis(sulfosuccinimidyl)suberat), which is used in the method Flow-FISH for the differentiation of cell surface antigens . Based on this method, it was hypothesized that the use of BS3 will contribute to the preservation of the vital dye in the cell after heating up to 80°C, which will allow researchers to simultaneously determine telomere length and the number of executed divisions in cultures of proliferating cells.
The aim of the invention is to provide a method of simultaneous determination of telomere length in cells with a known quantity of committed divisions.
The method consists in the fact that the cells differentiated by the number of completed divisions using the vital dye gradualism lower level is th fluorescence. Simultaneously with this, without preliminary separation of target proliferating cell population from the General population, use the flow-FISH (in situ hybridization) to determine the length of telomeres in cells, differentiated by the number of completed divisions and surface marker. Procedures to produce involving crosslinker amino Bis(sulfosuccinimidyl)suberat(BS3).
The method allows to determine the length of telomeres at the level of single cells using in situ hybridization, as well as to determine the number of divisions made this cell in vitro with subsequent analysis on a flow cytometer.
This makes the method more accessible, greatly increases the information content and performance, reduces research time and costs.
Description of the method with examples of specific implementations
The investigated cells stained with the vital dye CFSE (Molecular probes, USA) with a final concentration of 2µm in RPMI 1640 15 minutes, then washed in RPMI with 10% FCS (HyClone, UK). After that, cells are cultivated in vitro.
After cultivation, the cells washed in SFR with 0.1% BSA (Sigma, USA) (SR-BSA) and mark biotinylating antibodies against the target antigen. Incubated for 20 minutes, washed SFR-BSA and add streptavidin labeled with fluorochromes, and incubated for 20 minutes. After washing add a solution BS34 mm (Bis(sulfosuccnimidyl)suberat, Pierce, US) for 30 minutes at 37°C . Then add Tris (Sigma, USA) at a final concentration of 20 mm and incubated for 15 minutes and washed SFR-BSA. Sediment resuspended in 300 μl of hybridization solution consisting of 70% formamide (Sigma, USA), 20 mm Tris and 1% BSA. The PNA probe complementary telomeric DNA sequences and labeled fluorochromes (Bio-Synthesis, Inc., USA), added at a concentration of 0.3 ág/ml . In parallel, put the sample of control cells, which is used as a standard hybridization. The sample is heated for 10 minutes at 80°C and hybridized 3 hours at +20°C. the cells are Then transferred into tubes for cytometry, washed twice with a solution of 70% formamide and once SFR-BSA + 0.1% of Tween. Sediment resuspended in 0.5 ml SPR-BSA. Sample analysis is performed on a flow cytometer. According to the parameters of forward and side light scattering allocate region lymphocytes, which determine positive cells in the CD4 antigen on channel fluorescence used fluorochrome. From the positive region to build a histogram of the fluorescence signal of the vital dye CFSE, which will be visible peaks in the distribution of cells by the number of past divisions. This histogram peak with the highest fluorescence determines nepodelishsya cells, and with minimal fluorescence of cells committed to the maximum number of divisions. After selecting a population of cells with the correct number on the lines build a histogram of fluorescence fluorochrome SS3, which Machen PNA probe complementary telomeric DNA.
Telomere length in cells is defined as the average fluorescence of the cells on the channel flurochrome SS3 after hybridization in the presence of PNA probe minus background (autofluorescence) of cells that have passed the same conditions in the absence of PNA probe. The ratio of the average length of telomeres in cells to the indicator control cells reflects the average telomere length relative to control cells.
The following is an example illustrating this method of determining the relative average length of telomeres in proliferating cells.
Example. Determination of changes in telomere length with each new division in subpopulations of CD4+ and CD8+ human lymphocytes in vitro culture for 7 days in response to the activation of anti-D3 monoclonal antibodies.
From 6 donors 5 ml of peripheral blood stabilized with heparin, sterile centrifuged in the gradient ficoll-urografin for 20 minutes at 3 thousand rpm, after which the ring mononuclear cells are collected and washed twice with 5 ml of sterile buffered saline solution (SFR) for 5 minutes at 1 Tyson/min. the Precipitate resuspended in 1 ml SPR and count the number of cells in the cell Goryaeva.
The lymphocytes are stained with the vital dye CFSE and cultured in 24-hole tablets in CO2incubator with 5% CO27 with the current number of 2 million to 1 ml of complete medium (RPMI, FCS 10%), glutamine 0.3 mg/ml, gentamicin 50 μg/ml, Tienam 25 μg/ml) with the addition of anti-D3 antibody 1 μg/ml (Medispectra, Russia) and IL-2 100 u/ml (biotech, Russia). After 7 days the cells are harvested and carried out the in situ hybridization using mouse anti-CD4 and anti-CD8 biotinylated antibodies (Becton Dickinson, USA). The in situ hybridization is carried out with a PNA probe complementary telomeric DNA sequence (SSTA)3marked fluorochroman SS3 (Bio-Synthesis, Inc., USA) with a final concentration of 0.3 ág/ml . For the control cells were taken murine splenocytes line C57B L/6 (Kennel "Dawn", Tomsk). Analysis was performed on a flow cytometer FACS Calibur (Becton Dickinson, USA) using the software Cell QuestPRO(Becton Dickinson, USA). After the separation of the gate of lymphocytes by forward and side svetorasseyanie was isolated cells positive for CD4+ (or CD8+) antigen, which determined the number of peaks on the channel fluorescence of the vital dye CFSE (figure 1). It was determined that 7 days the cells had committed a maximum of 7 divisions. From each gate corresponding to a certain number of committed lymphocytes divisions, was determined average telomere length by determining the level of fluorescence channel luminosity probe PNA-SS3; was additionally measured the average telomere length in splenocytes, as control cells, using the same probe (figure 2). Were determined relative telomere length in nepodelishsya cells and cells held from 1 to 7 levels for subpopulations of CD4+ (CD8+) lymphocytes. From Figure 3 it can be seen that nepodelishsya cells have short telomeres, and at the division of the gradual lengthening of telomeres with the peak on the 3rd division for CD4+ lymphocytes and 4-5 divisions for CD8+ lymphocytes. It was determined that under cultivation in vitro after activation of lymphocytes anti-CD3 antibody is an lengthening of telomeres, with great value for CD8+ cells. The data obtained indicate a significant influence of activation and in vitro cultivation on the molecular changes in lymphocytes.
Thus, the obtained results indicate the possibility of applying the method for measuring the average length of telomeres in cells depending on the number of traversed them tick. A significant advantage of this method is the simultaneous separation of cells by the number of committed divisions, surface marker and define them in average telomere length.
Sources of information
The method of measuring the length of telomeres in cells, differentiated by the number of completed divisions using the vital dye gradualism lower level of fluorescence, using the method flowFISH (in situ hybridization) and subsequent analysis on a flow cytometer, characterized in that telomere length measured in proliferating cells the General population simultaneously with their differentiation according to the number of divisions and differentiation by surface marker, which is used crosslinkers amino Bis(sulfosuccinimidyl)suberat(BS3).
SUBSTANCE: sample is prepared: an additional weight of nanocarbon forms is dispersed in 1 ml of organic solvents with a degree of polarity smaller than that of water - dimethyl sulphoxide or ethanol. Then it is mixed and exposed to ultrasound for 30 minutes. The prepared nanocarbon suspension is transferred in an aqueous medium to the final concentration of the used solvent 2.5 %. The produced and control samples are added with a viable sensor recombinant luminescent Escherichia coli K12 strain with cloned luxCDABE genes of luminescent Photobacterium leiognathi system. It is followed by incubation for 60 - 180 minutes, measuring luminous intensity and evaluating optical properties of the analysed suspension simultaneously. A toxicity index (T) is calculated with evaluating an actual luminous intensity of the strain (Iact) in comparison with the control of the same concentration of the solvent, considering light absorbing properties of the analysed suspension (D) and an experimental luminescence level of the bacterial luminescent biosensor (Iexp).
EFFECT: invention allows providing higher accuracy and sensitivity of nanocarbon biotoxicity analysis ensured by the introduction of a correction value - the actual luminous intensity of the strain Iact, considering a common factor of emitted light distribution in the analysed suspension.
SUBSTANCE: method involves collecting dust samples with a sterile cotton wool wad from 10-20 cm2 of any surface or the sterile cotton wool filter of a vacuum cleaner. The dust samples are put into a sterile physiological solution, followed by extraction for 30-60 minutes at room temperature while stirring. The supernatant liquid is collected and multiple cultures of the obtained extract are prepared from the liquid. The obtained cultures are sown in a semi-liquid thioglycol medium and modified thioglycol medium obtained by adding defibrinated blood to the thioglycol medium in a given amount. Culturing is performed for 10 days at temperature 37°C and daily visual monitoring, followed by determining the number of microbial cells which are calculated from the McCready probability table, which is compiled based on data on growth properties of microorganisms and tinctorial properties of the bacterial community of the dust sample are determined through microscopic examination of smears of the mixed culture mass which is gram-stained.
EFFECT: simultaneous quantitative determination of the total bacteria number and obtaining cultural, morphological, tinctorial and haemolytic characteristics of the bacterial community of dust samples from buildings.
SUBSTANCE: biosensor contains photo-autotrophic microalgae cells, the fluorescent characteristics of the photosynthesis system of which vary in the presence of cytotoxic chemical compounds in their surroundings: heavy metal ions and herbicides. Cells of green and diatomic microalgae are immobilised in cryogenic gel of polyvinyl alcohol: a cell suspension is deposited on a surface and the cells enter macropores of the polymer carrier under the effect of centrifugal force (5000-14000 g) for 1-10 minutes. A highly sensitive and stable biosensor is obtained based on components taken in the following ratio in wt %: microalgae cells 0.015-1.1; polyvinyl alcohol 7-15; aqueous phase - up to 100. Low concentrations of heavy metals and herbicides in aqueous systems are determined at flow rate of up to 360 ml/h based on the change in the value of relative variable fluorescence chlorophyll cells in the biosensor. The biosensor can be used for a maximum of 60 days.
EFFECT: high sensitivity of the biosensor.
2 dwg, 5 ex
SUBSTANCE: detection and identification of biological objects and their nanocomponents are enabled by exposure to radiation, monochromatic or nonmonochromatic radiation, including laser, sensing of one or more samples containing microobjects and their nanokcomponents with the use of a set of sample response measurement and record devices. The responses characteristics of each radiation conversion event are measured separately or in the aggregate, transferred and reduce in a diagnostically linear form. It is followed normalisation, correction and creation of a base of the reference and diagnosed parametres of microobjects and/or their nanocomponents. Further, the reference, diagnosed and identified microobjects and/or their nanocomponents are recognised and compared with experience data of required parametres on the basis of measurement with the use of a matrix.
EFFECT: use of the declared method allows precise qualitative and quantitative analysis of detected, identified, diagnosed parametres of the microobjects and their nanocomponents on the basis of optical measurement.
4 cl, 12 dwg, 1 tbl, 3 ex
SUBSTANCE: cultivated microbiological objects count is ensured by the measurements of their morphological compositions by determining the size distribution of microorganism cells in a nutrient fluid by variation of scattered light intensity. The fluid flow is sounded with monochromatic coherent light; interaction signal of probe radiation and analysed microbiological objects are recorded by the measurement of amplitude and duration of scattered light pulses by analysed particles; and functions derived from the measurements are plotted in the form of two-dimensional distribution of specified amplitudes and durations expressing statistic parameters of light scattering intensity by particles. After said functions, the size distribution of analysed cultivated microbiological objects and decay products of the nutrient fluid is derived.
EFFECT: higher measurement accuracy due to eliminated error caused by foreign particles that are decay products of the nutrient fluid in cultivation of the analysed microbiological objects.
9 dwg, 5 ex
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
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.
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
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.
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: biology and medicine.
SUBSTANCE: invention relates to the method of improving stem cells mobilization; the essence of the invention lies in the application of hyaluronidase immobilized on the basis of low-molecular polyethyleneglycol 1500 Da by a flux of running electrons at the voltage of 2.5 MeV as a medium herewith strengthening the stem cells mobilization.
EFFECT: improved stem cells mobilization.
5 ex, 5 tbl
SUBSTANCE: cells of bone marrow are subjected to 30-minute pre-incubation in nutrient medium which contains 1 U/ml of hyaluronidase, and are twice washed by centrifugation. After that non-adhering fraction of myelocariocytes is cultivated in nutrient medium with addition of granulocytic colony-stimulating factor.
EFFECT: invention makes it possible to perform efficient stimulation of pluripotent hemopoetic stem cells in vitro.
1 tbl, 1 ex
SUBSTANCE: immunocytochemical estimation method for proliferative lymphocyte state by an expression behaviour of an C-terminal B23/nucleophosmin protein fragment in indirect immunofluorescence test provides a visual cell estimation for count and size of luminous nucleoli marked by a specific anti-peptide antibody to the C-terminal B23/nucleophosmin protein fragment detecting a B23.1 protein oligomer form only of molecular weight 210-230 kDa. The intact lymphocyte value found in a cycling state G0 is the presence in a cell nucleus of no more than one luminous nucleoli detected by the specified C-fragment B23 antibody, while 2-3 luminous nucleoli are detected in lymphocytes in the process of proliferative lymphocyte activity caused by PHA stimulation in 24 hours from the initiation of the stimulation procedure, and 4-5 luminous nucleoli of greater size and more intense luminescence between + to +++ as compared with the reference are detected in the cells in 48-72 hours from the initiation of the stimulation procedure when most lymphocytes are found at the peak of proliferative activity in the cycling state S.
EFFECT: invention enables early diagnostics of malignant haematological and oncological diseases.
3 dwg, 1 tbl, 2 ex
SUBSTANCE: method of human regulatory CD4+CD25+FOXP3+ T-cell enrichment ex vivo consists in recovery of CD4+ T-cell population from patient's peripheral blood and cultivation thereof in a growth medium containing 5-10 % autologous serum, 10-1000 units/ml of interleukin-2, 0.5-10 mcg/ml of monoclonal human CD3-antigen antibodies, 0.1-10 mcg/ml of monoclonal human CD28-antigen antibodies and 1-50 ng/ml of the transforming growth factor-beta1 per 1 ml of the growth medium.
EFFECT: invention allows producing the regulatory T-cell enriched autologous lymphocyte population suitable for treating the diseases characterised by prominently decreased T-cell count or functional activity.
10 dwg, 1 tbl
SUBSTANCE: what is described is a composition for fistula treatment in an individual, including stromal stem cells of fatty tissue where at least approximately 50 % of stromal stem cells of fatty tissue making a composition, express CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 markers and where the contents of the stromal stem cells of fatty tissue in the composition makes at least approximately 3×106 cells/ml.
EFFECT: invention extends the range of products for fistula treatment.
8 cl, 7 dwg, 4 ex
SUBSTANCE: method includes separation by centrifugation of heparinised mononuclear cells of umbilical blood (further UB) in gradient of density on Ficoll-Paque™PLUS. After that realised are thorough washing in PBS+0.1 % BSA medium and denaturation at temperature 82°C of suspension, consisting of UB cells and control cell culture 1301, supported in RPMI medium with addition of 10% bull serum of glutamine and penicillin with streptomycin. After that, analysed and control cells are distributed in two pairs of test tubes, adding to them up to 0.5 ml PBS 0.1% BSA solution, centrifuging with acceleration 500 g for 5 minutes. Then in two rest tubes with cells poured is hybridisation buffer, and into the second pair - poured is hybridisation buffer with peptide-nuclein probe. After that, they are incubated in darkness for 2-20 hours, washed, mixed, centrifuged, stained in solution of propidium-iodide and RNK A. After that, analysis of cell samples is carried out with determination of their relative length telomeres depending on length of telomeres of cell culture 1301, taking into account DNA index.
EFFECT: invention makes it possible to increase quality of determination of properties of transplant, samples of hemopoetic stem cells of umbilical blood for transplantation.
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed group of inventions relates to medicine, namely to neurosurgery and biotechnology, and can be used for cultivation of nerve stem cells of mammal, excluding embryonic human cells, and for treatment of spasticity, rigidity, spinal cord or amniotrophic state in subject, requiring such treatment. For cultivation of said cells preliminarily incubated in culture vessel is solution, which contains poly-D-lysine in concentration from 0.1 mkg/ml to 1 mg/ml during from 5 minutes to 3 hours. After that, culture vessel is rinsed and dried. Nerve stem cells are inoculated into said culture vessel without serum. After that, solution of fibronectin and, at least, one mitogen is added into culture vessel and nerve stem cells are cultivated without serum. Then, cultivated nerve stem cells are passed until fusion. For treatment of said states nerve stem cells, cultivated be claimed method, are concentrated. Therapeutically efficient amount of said concentrated nerve stem cells is introduced into region of patient's central nervous system tissue with decreased level of GABA-producing or glycin-producing neurons.
EFFECT: group of inventions ensures efficient method of cultivating mammalian nerve stem cells, excluding embryonic human cells, which makes it possible to achieve in vitro higher degree of neuronal differentiation to the level sufficient for treatment of said states and improvement of survival in vivo of such cells, as well as efficient treatment of spasticity, rigidity or amniotrophic state in subject due to introduction into their central nervous system tissues with lower level of GABA-producing or glycin-producing neurons of cells, able to differentiate into sufficient number of GABA-producing or glycin-producing neurons in said tissue.
10 dwg, 7 ex
SUBSTANCE: method includes filling work chamber with suspension of tumour cells and their aggregates. After that carried out is complex processing of suspension of tumour cells and their aggregates by high-amplitude low-frequency ultrasound and ozone/NO - containing gas mixture, introduced immediately into volume of insonifies and bubbled suspension, as well as ozone/NO-containing solution, which is formed at barbotage of suspension with ozone/NO-containing gas mixture.
EFFECT: invention makes it possible to increase efficiency of process of disintegration of suspension of tumour cells and their aggregates for maximally possible separation from them of biologically active substances.
6 dwg, 1 ex
SUBSTANCE: method of cell expansion includes culturing adhesive mesenchymal stromal cells from placenta or adipose tissue in three-dimensional conditions of culturing, which contribute to expansion of cells.
EFFECT: invention allows obtaining adhesive mesenchymal stromal cells of placenta or adipose tissues, which can by applied for supporting hematopoetic stem cells, for immunosuppression, for obtaining conditioned media in transplantation.
22 cl, 22 dwg, 3 tbl, 3 ex
SUBSTANCE: method includes opening of abdominal cavity, cannulating of v.portae, through which two-step perfusing is performed, mechanical processing of liver, purification of hepatocytes, filtration and differential centrifuging, preliminary incubation and inoculation of hepatocytes in Goryaev chamber, primary perfusing being performed with calcium-free Hanks solution, the secondary one being performed three-four times with Hanks solution, which contains calcium ions and 1 type collagenase in concentration 0.02-0.05% for 20 minutes at perfusate temperature 15-16°C; purification of hepatocytes in ficoll gradient is performed three-four times for 15 minutes on centrifuge with 1000 rev/min at temperature 20°C with addition of 0.5-1.15 M of saccharose, differential centrifuging is carried out with 700 rev/min with 2 minute exposition; preliminary incubation of hepatocytes is carried out at 37°C for 20 minutes.
EFFECT: invention makes it possible to increase percentage of output of viable hepatocytes of birds of genus Columba livia, and can be used for studying mechanisms of cell functioning, in diagnostics of viral infections, in production of vaccines, correction of functions of injured liver tissues.
3 tbl, 3 ex
SUBSTANCE: to 1 part of water solution, which contains 1 mg/ml of plasmid DNA, added are 10 parts of water and 1 part of suspension, containing 4 g/ml of superparamagnetic parts of magnetite. After that into obtained solution added is 1 part of buffer solution with pH 7.5, 2 parts of 0.005 M solution of ribonucleotidetriphpsphates, 2 parts of 0.1 M of dithiotreitole, 1 part of solution, containing 2 mg/ml of bovine serum albumin, 1 part of solution, which contains 10 units of RNAase inhibitor and 1 part of solution, which contains 10 units of PNA-polymerase of T3 or T7 or SP6 bacteriophage. Obtained mixture is incubated for not less than 1 hour at temperature +37°C and placed into magnetic field with intensity not less than 0.2 T for 1-3 minutes. After that water phase, containing target RNA is decanted.
EFFECT: RNA, obtained by claimed method, is of high quality and is suitable for molecular-biological studies of gene structure and functions, as well as for diagnostic and therapeutic purposes in medical practice.
2 cl, 1 dwg, 1 ex