Method to extract stem cells from bone marrow for intravascular introduction

FIELD: biotechnologies.

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

The number of mononuclear cells (1 l)
Patient N. and/b No. 17140 with/aboutThe placeholderThe proposed method
The number of nucleated cells (1 l)of 18.2×10928,4×109
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 suspension5,5×10613×106
Viable96%98%
Patient p and/b No. 18131 with/aboutthe placeholderThe proposed method
The number of nucleated cells (1 l)9,36×10910,1×109
of 1.05×109(11,23%)1,28×109(12,76%)
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 suspensionof 1.5×106of 3.3×106
Viable95%100%

CLINICAL EXAMPLES.

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.



 

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1 ex, 5 dwg

FIELD: medicine.

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

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