Method to extract stem cells from bone marrow for intravascular introduction
SUBSTANCE: method is proposed to extract stem cells, including whirling of heparinised bone marrow with hydroxyethyl starch at the ratio of source ingredients of 1:2 with speed of 700g for 15 min. in the closed system of three haematological containers connected to each other with tubes with subsequent removal of fat admixtures and plasma into the container No.1, transfer of the mononuclear fraction of bone marrow, a part of supernatant and erythrocytes adjoining the interface of two media into the container No.2. Sludged erythrocytes and bone fragments remain in the main container, whirling of the produced sample with the speed of 900g for 15 min. in the container No.2 to produce cell material for intravascular introduction, at the same time after the specified whirling a part of supernatant is removed into the container No.1 without unsealing of the system.
EFFECT: production of paracrine effect of bone marrow mononuclear cells and provision of safety.
1 tbl, 2 ex
The invention relates to regenerative therapy in cardiology, in particular to the development of techniques for the separation of the bone marrow to maximize the number of viable stem cells with the aim of intravascular injection for regeneration of the body.
The allocation of the mononuclear fraction of bone marrow (MPCM) for experimental purposes has been known since the 1960s, It is in MPCM detected all known up to the present time human stem cells, namely: hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, and others. In recent years MPCM was used for regenerative therapy primarily in cardiology. Since 2002 the German cardiology (Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circ 2002; 106:1913-1918 Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circ 2002; 106:1913-1918) began to apply MPCM for the treatment of cardiac patients, mainly in myocardial infarction.
The first study to assess the regenerative therapy using MPCM conducted in 2004 in Germany. - TOPCARE-AMI (Transplantation Of Progenitor Cells And Regeneration Enhancement in Acute Myocardial Infarction) (2001-2003, Germany). The results of the study showed the usefulness of MPCM in the treatment of acute myocardial infarction: in patients Ulu is sales fraction of exile, better restore the work of the left ventricle of the heart. In 2010 published the results of a large controlled study of the STAR-heart study: autologous bone marrow mononuclear cells obtained by gradient centrifugation with picollo, was administered to patients with heart failure with a fraction of the expulsion of 35% or less. After 5 years there has been an improvement of contractility and survival in the study group compared with the control (conservative treatment without injection of mononuclear cells) (B.E. Strauer, Yousef M., Schannwell C.M. The acute and long-term effects of intracoronary Stem cell Transplantation in 191 patients with chronic heart failure: the STAR-heart study. European Journal of Heart Failure 2010. - V12. - p.721-729).
2011 has already conducted more than 30 pilot and randomized, placebo-controlled studies. The most significant of them: MAGIC BOOST, TOPCARE-AMI, REPAIR-AMI, ASTAMI, STAR-heart and others. The results of these studies overwhelmingly demonstrate the benefit of applying MPCM for the treatment of cardiac patients. Almost all the works devoted to cell therapy in cardiology, selection MPCM was conducted using ficoll.
The known method of obtaining stem cells using magnetic sorting, which allows you to obtain a separate pools of stem cells, which are monoclonal antibodies fixed on them by the iron atoms. Magnet the initial sorting allows you to get the most pure pools of cells, what is important in research work. However, for patients with severe organ damage, such as the heart, it is important to get the maximum number of stem cells that will ensure the regeneration not only of cardiomyocytes and vessels conducting system of the heart, of the elements of the stroma and extracellular matrix. It is therefore important getting the entire spectrum of stem cells involved in regeneration. Moreover, the earliest stem cells may not have surface markers, or they are unknown, therefore, these stem cells cannot be allocated by the method of magnetic sorting.
There is a method of allocating MPCM using gelatin, which involves primary sedimentation of erythrocytes using these substances for 1.5-3 hours without centrifugation before the formation of platelet-leukocyte film (instruction on identification, conservation and use of leukocyte mass. Moscow, 1969), which significantly increases the time of the final selection MFCM and makes it almost impossible to carry out a simultaneous revascularization of the myocardium and the introduction of autologous bone marrow cells (MPCM) during one procedure. In addition, a solution of gelatin is a protein product of animal origin, which are described anaphylactic reactions. (B.A. Baryshev. The blood substitutes. Src is wochnik for physicians. SPb. 2001. - p.15-19).
Application methods-analogues, including with the use of HES for selection MFCM, revealed significant shortcomings of these methods in the form of large losses of different pools of stem cells. Some stem cells, including hematopoietic, determined among red blood cells, which are located at different levels under leukocyte-lymphocytic film. This part, according to our data, ranges from 25 to 50% of all hematopoietic stem cells and it is this part of the cells is removed together with erythrocytes in all ways-analogues and the prototype method of obtaining stem cells. All of the above methods-analogues and prototype method was used to prepare the stem cells of the bone marrow for cryopreservation with subsequent long-term storage until the time of request. Therefore, the main requirement was maximum destruction of red blood cells, because they dramatically affect the results of the cryo-storage of samples of bone marrow. However, if allocated stem cells are supposed not to preserve, but to use the same patient immediately after their selection vnutrisosudisto, the degree of removal of erythrocytes from a mixture MFCM is not fundamental, and located among the red blood cells stem cells with active adhesion molecules on their surface, prob the Dublin core through the microcirculation of the affected organ, effectively fixed to the endothelium of blood vessels that penetrate the tissues and provide further its regeneration. The volume of plasma that remains in the proposed method, together with MFCM and part of the red blood cells will be much more than a how-analogues and method-prototype, since the resulting suspension of cells injected vnutrisosudisto. Fundamental to this method of introduction is complete removal of fat, bone fragments and blood clots, as this may cause a fatal embolism peripheral channel vessels.
The closest in technical essence is the allocation of the mononuclear fraction of the blood and bone marrow by the method of Boyum (Boyum A. Separation of leukocytes from blood and bone marrow // Scand. J. Clin. Lab. Investig. - 1968. - Vol.21 - Suppl.97, p.1-9). The principle of the method is based on the difference in buoyant density of the formed elements of blood. The mixture of the polysaccharide ficoll and radiopaque substance isopach or urografin creates a density gradient, which allows centrifugation to separate cells from peripheral blood and bone marrow mononuclear cell fraction, which includes lymphocytes, monocytes and stem cells, and the fraction containing granulocytes and erythrocytes. Cells of the mononuclear fraction is smaller than ficoll, density and are located above it. The density of erythrocytes more and they pass through ficoll, falling on D. what about the tubes.
Methods: mononuclear cell fraction isolated from heparinized bone marrow (20 IU of heparin/ml for bone marrow). Bone marrow is diluted with a saline solution (pH of 7.2) at a ratio of 1:5. Diluted bone marrow is centrifuged in a density gradient ficoll-urografin, the density of 1.077 g/ml (Kheifets LB, Abalkin, VA separation of the formed elements of human blood in the density gradient urografin-ficoll // lab. business, 1973. - 10. - S-581) at 400g for 30 min in a special test tube with a stopper. Obtained by sampling with a sterile pipette mononuclear fraction washed three times in SFFR (saline buffered with phosphate-saline buffer) and resuspended in medium RPMI-1640 (Flow Laboratories, UK) at a concentration of 106cells in 1 ml of cell Viability, determined by the method of staining with Trifanova blue, does not exceed 96%. To ensure the sterility of such an open system all manipulations with the cellular material is carried out in specially equipped clean room.
The disadvantages of this method include the following.
1. Cannot adhere to complete sterility, taking into account the leakage of the system.
Source this method was proposed for laboratory research. In the ASTAMI study was 1 case of sepsis due to infected material in the process of allocating AMCM.
. The use of toxic reagents forces three times to wash the obtained MPCM in saline solution before use in patients that do not warrant full-laundering and does not guarantee the absence of negative influence of toxic substances on the potential of stem cells.
3. At the same time at wash removes useful products of stem cells in the bone marrow growth factors, cytokines, chemokines, which play a positive role for tissue regeneration.
4. Selected using ficoll stem cells produce colony forming units in the environment of alpha-MEM worse than stem cells in other ways, i.e. they lose some of their potential, which is important for regenerative therapy.
5. Rather time-consuming method; on the selection of stem cells takes 3 to 5 hours and requires experienced personnel, as even minimal disruption of the technology selection leads to the loss of almost all stem cells.
The present invention is to develop a method of allocating the maximum number of viable stem cells from the bone marrow in a closed (sterile) system without the use of toxic substances for intravascular introduction of the received cellular material.
The developed method provides the use of a paracrine effect of mononuclear cells from bone marrow and safety of intravascular injection received cellular material.
In the proposed method does not use toxic substances to highlight MFCM. Gidroxiatilkrahmal is one of the widely used products (9).
All growth factors, cytokines and chemokines produced by cells present in the mononuclear fraction, when using the proposed method to continue to be entered together with the received cellular material. The viability of stem cells in the proposed method is 98% or higher, which also indicates the most favorable mode of hematopoietic stem cells (in the prototype, this figure is no higher than 96%). The application of the proposed method of obtaining stem cells from bone marrow for intravascular injection avoids loss of stem cells or they are minimized. The system remains closed during the entire period allocation MPCM that provides complete sterility at all stages of the selection.
The method consists in the following.
Bone marrow is obtained in the operating room with a puncture flat bones (sternum, iliac coast is th) in a volume of 140 ml, stabilized with heparin solution (15-30 IU per 1 ml bone marrow in sterile conditions is introduced into the main plastic Hematology container is connected by a tube with two containers No. 1 and No. 2; adds gidroxiatilkrahmal of 1:2. The system is sealed in the operating room. At the first stage of centrifugation is specified suspension with acceleration 700g for 15 min, and then using fractionator medical blood components is the removal of impurities fat and plasma in the container 1 and MFCM, part of the supernatant and cells, adjacent to the line of division 2 environments, is placed into the container-satellite No. 2. Mainly the container remain ladirovannye erythrocytes and bone fragments. In the second stage for the reduction of the received amount spend a second centrifugation of the cell suspension mode 900g for 15 min to obtain a final volume of suspension (60 ml)after centrifugation to remove the portion of the supernatant into a container No. 1. Thus, from the moment of exfuze bone marrow and sealing him in the operating room until the introduction of the cellular material in the bloodstream of the patient is maintained sterility of the obtained sample. At the end of the extraction procedure are blocked (sealed) tube that connects the main container of containers No. 1 and No.. For intravascular injection is used cellular material from container No. 2.
An EXAMPLE of the METHOD
Bone marrow is obtained in the operating room with a puncture flat bones (sternum, iliac bones) in a volume of 140 ml, stabilized with heparin solution (25-30 units per 1 ml bone marrow), is introduced into the main plastic Hematology container is connected by a tube with two containers No. 1 and No. 2; adds 6% gidroxiatilkrahmal of 1:2 (1 part hydroxyethylamine and 2 parts bone marrow). The system is sealed in the operating room. Is the centrifugation of the specified suspension with acceleration 700g for 15 min, and then using fractionator medical blood components is the removal of impurities fat and plasma in the container 1 and the mononuclear fraction of the bone marrow, the part of the supernatant and cells, adjacent to the line of separation of two media, translated into container No. 2, mainly container remain ladirovannye erythrocytes and bone fragments. For the final selection MFCM, leaving the required volume of plasma (60 ml) need a second centrifugation of the cell suspension mode 900g for 15 minutes At the end of the extraction procedure are blocked (sealed) tube that connects the main container with the container. For research cleto the surface of the obtained material part of it (2 ml) can be separately sealed in the nearest part of one of the connecting tubes. Thus, the control sample may be taken for research without opening the main container, which is an additional positive element of the proposed method. For intravascular injection is used cellular material from container No. 2. A method of allocating MPCM only suitable for intravascular introduction in any organ with autologous use: in the heart, liver, kidneys. Not only intra-arterial route of administration, but also intravenously, for example, through the umbilical vein into the system of the portal vein of the liver.
Limitations: this method is not suitable for the purposes of preservation of stem cells, for intramyocardial introducing the resulting suspension MFCM, for the purposes of allogeneic transplantation.
Estimated number of nucleated cells, the number of mononuclear cells, the number of hematopoietic stem cells (CD34+) using flow cytofluorimetry in the samples in the comparative selection of mononuclear cells by using the proposed method and the prototype method showed comparable results, with performance by lymphocytosis, the number of mononuclear cells and the number of hematopoietic cells were better than the proposed method (table 1), while the time spent on the selection MFCM, using the prototype method was 3 to 4 h, and using PR is degenova way - 45 minutes
|Patient N. and/b No. 17140 with/about||The placeholder||The proposed method|
|The number of nucleated cells (1 l)||of 18.2×109||28,4×109|
|The number of mononuclear cells (1 l)||to 3.36×109(18,5%)||6,5×109(22,8%)|
|The number of CD 34+(1 l)||of 1.1×107(0,6%)||of 2.6×107(0,8%)|
|The absolute number of CD34+ cell in 50 ml suspension||5,5×106||13×106|
|Patient p and/b No. 18131 with/about||the placeholder||The proposed method|
|The number of nucleated cells (1 l)||9,36×109||10,1×109|
|The number of CD34+ (1 l)||of 3.1×107(0,3%)||6,6×107(0,61%)|
|The absolute number of CD34+ cell in 50 ml suspension||of 1.5×106||of 3.3×106|
Example 1. Patient C. 68 years (and a/b No. 17821 C/o). In 2001, the patient underwent surgery: coronary artery bypass grafting. In 2004, because of the recurrence of angina was performed intracoronary transplantation MPCM for the purpose of regeneration of terminal vascular beds of the coronary arteries. Selection MPCM was carried out as described above. After 1 year marked a distinct decrease the zone of insufficient blood supply of the left ventricle according to single photon emission computed tomography (SPECT); this was a distinct improvement in clinical condition: reduced the number of angina attacks voltage, disappeared angina at rest, improved exercise tolerance. PA is jent moved from IV in II functional class of angina voltage. The improvement continues during 3 years of observation.
Example 2. Patient railway History No. 1831 C/o 2004 Suffered from coronary heart disease within 5 years. During hospitalization had III functional class of angina voltage. When SPECT identified area of hypoperfusion of the myocardium. When coronary angiography detected occlusion of the circumflex coronary artery. To perform coronary angioplasty is impossible due to occlusion over a large area. During coronary angiography performed introduction MPCM in the left coronary artery (40 ml) and 20 ml in the right coronary artery. Procedure the patient underwent fine. Any change therapy or other treatments was not. After 6 months. significantly decreased angina voltage, they only occur after vigorous exercise. Control SPECT revealed a significant improvement of myocardial perfusion.
To September 1, 2011 170 patients underwent introduction MPCM in the coronary bed. There were no complications. Clinically positive result was confirmed by reduction of the functional class of angina and heart failure note 97% of patients. Instrumental confirmation of the reduction of area of critical ischemia of the myocardium in the assessment using positron emission tomography and single photon emission computed tomography for observing in speaker is obtained in 90% of patients. All patients with chronic heart failure by echocardiography showed improvement of contractility. These data show that the effect of intravascular injection MPCM associated with the intensification of the processes of circulation and regeneration in the myocardium. After 1 year, the positive effect is preserved in 80% of patients after 5 years - more than 60% of patients.
The method of obtaining stem cells from bone marrow for intravascular administration, including centrifugation of heparinized bone marrow, characterized in that the selection of stem cells is carried out in two stages, the first stage is implemented by centrifugation of heparinized bone marrow with hydroxyethylcellulose in the ratio of initial components 1:2 speed 700g for 15 min, and the centrifugation is carried out in a closed system of three hematological containers connected by a tube with the subsequent removal of impurities fat and plasma in the container No. 1, mononuclear fraction of bone marrow, the part of the supernatant and cells, adjacent to the line of separation of two media, transferred to container No. 2, mainly container remain ladirovannye erythrocytes and bone fragments, the second stage is carried out by centrifugation of the obtained sample with the speed 900g for 15 min in which ontainer No. 2 to obtain cellular material for intravascular injection, when referred to centrifugation to remove the portion of the supernatant into a container No. 1 without depressurization system.
SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.
EFFECT: possibility to use in diagnostics of virus infections.
SUBSTANCE: method is proposed to cultivate human and animal cells on nutrient medium containing serum of buffalo cow foetus blood in the volume ratio of 5-7% serum and 93-95% medium, with the following incubation of cultures at 37°C for 3-5 days until formation of the monolayer.
EFFECT: invention may be used for cultivation of cells in a culture.
SUBSTANCE: method is proposed to treat experimental pulmonary tuberculosis in mice using transplantation of stem cells by introduction of a suspension of stem cells resistant to tuberculosis with "k" genotype in the area H-2E into a caudal vein once a week.
EFFECT: method improvement.
SUBSTANCE: means comprises at least one phenolic compound from the group of derivatives of cinnamic acid or a mixture of such compounds, and a nonionogenic surfactant or a mixture of such substances in an amount of not less than 75% by weight.
EFFECT: invention enables to obtain beauty products, biologically active additives and food products on the basis of the described means and to use them to stimulate the reparative processes in a cell and to reduce the side effects of aggressive cosmetic procedures.
30 cl, 7 dwg, 3 tbl, 38 ex
SUBSTANCE: population of Tr1 cells is identified, being aimed against food antigen from regular ration of a human being and having phenotype CD4+CD25-FoxP3- in idle condition. The produced population of Tr1 cells in a combination with one or several pharmaceutically acceptable carriers, or in a combination with one or more pharmaceutical agents, used to treat inflammatory bowel conditions and selected from a group including anti-TNF, natalizumab, anti-IL1, anti-IL-6, anti-IL-12, anti-IL-17 and anti-IL-23, analogues of antagonist of a receptor IL-1.5 aminosalicylic acid and its analogues, corticoids, probiotics, methotrexate, azathioprine, 6-mercaptopurine, thalidomide, leflunomide, used within pharmaceutical compositions for treatment of inflammatory bowel conditions.
EFFECT: invention makes it possible to produce an efficient agent for treatment of inflammatory bowel conditions.
15 cl, 8 dwg, 1 tbl, 2 ex
SUBSTANCE: permanent line of cells is produced from tissue of trout gonads by means of long-term passage in the medium of Igla MEM on Earl salts with 25 mM of HEPES from 10% embryonic serum. The line may be used as a test culture for extraction, accumulation, titration and study of a infectious pancreas necrosis virus (IPNV) of salmon fishers, virulent haemorrhagic septicemia virus (VHSV) of salmon fishes, infectious hemopoietic necrosis virus (IHNV) of salmon fish tissue, spring viraemia of carps virus (SVCV), koi herpes virus (KHV), sturgeon herpes virus (SbSHV).
EFFECT: permanent line of OMG cells will find wide application in laboratory research on virosology and biotechnology in production of virus vaccines and diagnosticums.
SUBSTANCE: patient's peripheral blood is simultaneously examined for the percentage of circulating stem hemopoietic cell CD34+ (SHC) and the volume fraction of lymphocytes (Lph, mln/ml); they are individually related as R by formula R=LPH/SHC, and an R (y axis) to SHC (x axis) law curve is drawn to be used as a calibration curve within the log-log grid, while the current recipient's state is assessed by examining the peripheral blood for LPH and SHC to be related as Rp and found in the calibration system of axis. If Rp is found either on the calibration curve, or above the same, the current recipient's state is considered to be favourable, otherwise the unfavourable state is shown by Rp found under the calibration curve.
EFFECT: using the declared method enables improving the accuracy of assessing the post-transplantation recipient's state.
4 tbl, 4 dwg, 3 ex
SUBSTANCE: method is described to stimulate expansion of haematopoietic stem cells (HSC) in vitro or in vivo. A population of cells is incubated, which contains HSC, in presence of at least one HSC modulator, at the same time the HSC modulator is selected from the group that consists of PGE2, PGI2, linoleic acid, 13(s)- HODE, L Y 171883, MIDA acid, eicosatrienoic acid, epoxyeicosatrienoic acid, ONO-259, Cay 1039, an agonist of receptor PGE2, 16,16-dimethyl-PCE2, 19(R)-hydroxy-PCE2, n-(n-acetamide benzamide)phenyl esther 16,16-dimethyl-PCE2, 11-desoxy-16,16-dimethyl-PCE2, 9-desoxy-9-methylene-16,16-dimethyl-PCE2, 9-desoxy-9-methylene-POE2, butaprost, sulprostone, serynolamide PGE2, methyl ester PGE2, 16-phenyltetranor-POE2, 15(S)-15-MeTnn-PGE2 and 15(R)-15-MeTnn-PGE2. A method is proposed to strength HSC acceptance, and also a method to strengthen recovery of haematopoietic stem cells, which include HSC incubation in presence of at least one HSC modulator.
EFFECT: invention expands arsenal of methods for stimulation of growth of haematopoietic stem cells in a subject.
28 cl, 24 dwg, 11 tbl, 2 ex
SUBSTANCE: by the recombinant method a line of cells CHO[V3D] is produced, which are transformed with plasmid pV3D, coding the human vessel endothelium growth factor, isoform A165, with C-terminal 3xDED-epitope (SEQ ID NO: 1), which is able to produce a biologically active recombinant protein VEGF-A165 of humans with C-terminal 3xDED-epitope in the amount of at least 0.2 mg 1 l of medium for 72 hours with 1 ml of growth medium per 5 cm2 of single-layer culture. The cell line is deposited in the All-Russian Collection of Industrial Microorganisms VKPM under the registration number VKPM H-123.
EFFECT: no impact at biological activity of protein and possibility to do protein treatment.
4 dwg, 7 ex
SUBSTANCE: method comprises adding to the part of the wells with lymphocytes of mitogens or test substances, incubation of the contents of the wells, selection of the contents of the wells, cytolysis and isolation of nucleic acids. The reaction of reverse transcription (RT) is carried out with primers for genes tpa and cdc2: primer for RT with mRNA of gene tpa 5'-CATCTTCATCTCACTCTTC-3', primer for RT with mRNA of gene cdc2 5'-CTGGAGTTGAGTAACGAG-3'. Real-time PCR is carried out with the obtained cDNA on the specific primers and with use of the labeled oligonucleotides for genes tpa and cdc2: for gene tpa the forward primer is 5'-TGTCGTGTCAGACCTTGAAGC-3', the reverse primer is 5'-CCTTGGATTTCTTGCTTGTGAC-3', the probe (FAM)-5'-TGTACCACTGTCTCAAGCCCTCCTGC-3'-(BHQ1), for cdc2 gene the forward primer is 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', the reverse primer is 5'-CCAGAGTGTTACTACCTCATGTG-3', the probe (FAM)-5'-TGCCTTGCCAGAGC(FdT)TTTGGAATAC-3'-(BHQ1). The degree of proliferation of lymphocytes is expressed in proliferation index which is the number indicating how many times the amount of mRNA of gene tpa and/or cdc2 increases during culturing lymphocytes with/without addition of PHA or the test substance as compared with the amount of mRNA of the genes in lymphocytes freshly isolated from the blood of healthy donor.
EFFECT: invention enables to monitor directly the number of dividing cells, assessing the degree of expression of the genes involved and regulating the cell division.
4 dwg, 4 ex
SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.
EFFECT: possibility to use in diagnostics of virus infections.
SUBSTANCE: invention refers to medicine and veterinary science; it is used in transplantology, traumatology, surgery and oncology, and may be used to fill bone defects. What is described is a bioimplant which represents a donor bone deimmunised with chlorine-containing oxidants, a surface of which is covered with a multifunctional bioactive nanostructured coating (MBNC) of M-Ca-P-C-O-N or M-Ca-CON, wherein M is a metal specified in a group consisting of Si, Ti, Zr, Hf, Nb, Ta, and colonised by recipient's mesenchymal stem cells (MSC).
EFFECT: MBNC-coated bioimplant is in line with the anatomical and morphological characteristics of the replaced bone, provides cell adhesion, no transplant rejection, accelerated formation of connective tissue and callus.
8 dwg, 1 ex
SUBSTANCE: invention refers to medicine and veterinary science, namely to reconstructive surgery it aims at the applications in transplantation, traumatology, surgery, and oncology. What is described is a method for producing bioengineered constructs for bone defect replacement, which is based on a bone anatomically compatible with the replaced one which is deimmunised in 5-10% solution prepared of a dry mixture of sodium chlorite, sodium perchlorate, sodium chloride in a ratio of 7:2:1 and distilled water; it is coated with heterogeneous implantable gel and colonised with multipotent mesenchymal stromal cells recovered from the recipient's bone marrow using immunomagnetic separation technique.
EFFECT: method provides sizeable bone defect replacement, high strength, fast fixation and repair of the construct in the implantation region, causes no reject phenomena.
1 tbl, 4 dwg
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
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to chemistry of high-molecular compounds, namely to preparing film and spongy chitosan and collagen materials effective in human skin cell culture and wound grafting. A method for preparing the composite chitosan and collagen resorbable matrixes for human skin cell culture involves preparation of solutions polysaccharide chitosan and protein collagen, mixing them in preset proportions and formation of film and spongy matrixes of mixed polymer solutions. For this purpose, chitosan and collagen solutions in the concentration 1.0-4.0 wt % in a general solvent (aqueous 2 % acetic acid) are prepared, mixed in preset proportions, and the film and spongy matrix materials are formed from the prepared solutions. An amount of collagen in polymer mixtures makes 2.5-10 wt % (of chitosan). Further, films and sponges are heated to temperatures within 50-100°C for 1.0-5.0 h in an atmospheric environment.
EFFECT: use of the declared method allows preparing the film and spongy resorbable composite materials of natural polymers effective in human skin cell culture.
2 cl, 6 ex, 2 tbl
SUBSTANCE: invention refers to medicine, namely to cardiovascular surgery. A biological material for cardiovascular surgery is made of a preserved carp swim bladder.
EFFECT: reduced calcification with maintained physical-mechanical properties.
SUBSTANCE: invention relates to medicine, namely to cardiovascular surgery. Described is biological material for prostheses, in which Glisson's capsule of horse is used. As base, Glisson's capsule of horse is used.
EFFECT: increase of elastic-strength characteristics of prostheses.
SUBSTANCE: group of inventions relates to medicine, namely to dentistry, and deals with medications for restoration of periodontal tissues and methods of their obtaining. Biotransplant for treatment of periodontal diseases represents suspension which contains cultures of autological or allogenic fibroblasts, or fibroblast-like cells in 0.9% solution of sodium chloride in concentration 2.0-10.0x106 in 1 ml of biotransplant, integrated on pharmaceutically acceptable biocompatible biodegradable carrier, selected from the group: acellular matrix, hyaluronic acid preparation, acellular matrix and hyaluronic acid preparation, with specified volume ratio of cell culture suspension ands carrier, which makes it possible to obtain consistence convenient for injecting and, together with expressed clinical effect ensures low traumaticity and simplicity of injecting. Preferable, as carrier, used is crushed to size 100-200 mcm acellular matrix "Saimetra". Also claimed is method of treating periodontal diseases, which lies in the following: on mucous membrane of gum in region of defect one week before biotransplant introduction preliminarily made are cuts provoking an acute inflammatory process resulting in increase of tissue volume, which makes it possible to carry out lossless introduction of large volume of biotransplant. After that, into defect area by means of injection two times with interval 1-4 weeks introduced is claimed biotransplant, using 3.6-60×106 cells per treatment course, which ensures optimal time interval for cell engraftment and restoration of periodontal tissues.
EFFECT: increase of treatment efficiency.
7 cl, 3 ex, 2 dwg
SUBSTANCE: invention refers to medicine, and concerns a bioengineered structure for bony defect closure and osteogenesis which represents a hybrid implant comprising a porous polytetrafluorethylene membrane with a M-Ca-P-C-O-N or M-Ca-C-O-N doped multifunction, biocompatible, non-resorbed coating MBNC, where M is a metal chosen from a line including Ti, Zr, Hf, Nb, Ta, with autogenous or allogenic stromal cells recovered from fatty tissue or bone marrow passed on the surface thereof.
EFFECT: invention provides reliable degree of cell population attachment to the surface of the bioengineered structure with high cell culture adhesion and growth.
4 cl, 2 dwg
SUBSTANCE: invention refers to medicine, namely to traumatology and orthopaedics, and can be used for correction of dystrophic and traumatic changes of cartilaginous tissue, and for early correction of growth processes in idiopathic scoliosis. The method is characterised by the fact that growth plates of newborn miniature pigs are placed in Hanks solution with cannomycin 1 g/l for 5 minutes, then growth plates are washed, crushed, transferred to test tubes with RPMI-1640 medium with 20% FBS and 1.5% collagenase of activity 240 U/ml added, and incubated; the suspension is centrifuged at 2000 rpm for 10 minutes; isolated chondroblasts are cultivated upon concentration 50-60 million; cell passage is enabled with mixed 0.25% trypsin and 0.02% EDTA; further, the cells are centrifuged at 2000 rpm for 10 minutes; then, a cell aggregate is moved to a well with nutrient medium RPMI-1640 with 10% FBS added, and cultivated for 4-6 weeks to prepare a chondral graft of 2×2×2ħmm3.
EFFECT: invention prevents progressive spinal deformity at early stages, development of osteoarthrosis and osteochondrosis, reduced the postsurgical period, severely reduces number of postsurgical patient days and eliminates the staged expensive procedures on spine and joints.
1 ex, 5 dwg
SUBSTANCE: group of inventions refers to medicine. A method for preparing a biomaterial, which provides bone tissue regeneration, contains biphasic calcium phosphate (BCP) in the form of granules homogeneously dispersed in a three-dimensional blood protein mesh or bone marrow protein mesh, including the following steps: (i) mixing biphasic calcium phosphate in the form of granules of 40 to 500 mcm with blood or bone marrow aspirate in ratio 10 to 90 wt % of BCP of blood or bone marrow volume, g/ml; (ii) adding at least one coagulant to the mixture prepared at the stage (I) in an amount adequate to cause blood or bone marrow coagulation. The biomaterial further contains one additive specified in polymers, ceramics particles, pharmaceutical compounds, natural or synthetic growth factors, biomarkers, contrast agents, tissue or cell preparations. The kit for implementing the method comprises (a) a device having an internal sterile container with BCP; (b) a sterile container with a coagulant. The biomaterial is used in vitro or ex vivo as a carrier for produced bone tissue, or for producing a bone graft.
EFFECT: group of inventions provides bone tissue regeneration.
24 cl, 10 dwg