Method for obtaining autologous bone marrow cell concentrate

Abstract

The invention relates to medicine. A method for obtaining autologous bone marrow cell concentrate includes harvesting bone marrow aspirate, then centrifuging same and isolating a fraction containing mesenchymal cells. The bone marrow aspirate is harvested from the posterior iliac spine. The harvested aspirate is then introduced into the working chamber of a centrifuge and subjected to centrifugation, wherein during centrifugation, beams of light with different wavelengths are shined at the aspirate and the light beams passing though the aspirate are registered. At the same time, the following are removed successively from the working chamber of the centrifuge according to the wavelengths of the light beams: a fraction containing platelet-rich plasma, a fraction containing platelet-poor plasma, and an erythrocyte precipitate. The platelet-rich plasma is then diluted with excess platelet-poor plasma and filtered.

Description

[0001] METHOD FOR OBTAINING AN AUTOLOGOUS BONE MARROW CELL CONCENTRATE

[0002] DESCRIPTION OF THE INVENTION

[0003] AREA OF TECHNOLOGY

[0004] The invention relates to medicine, namely to methods for obtaining a biological preparation based on bone marrow concentrate and can be used in traumatology and orthopedics, neurology, in particular in the treatment of hernias and protrusions of intervertebral discs of the spine, in cosmetology, immunology and other areas.

[0005] LEVEL OF TECHNOLOGY

[0006] A method for isolating human bone marrow mononuclear cells is known, disclosed in Russian Federation Patent for Invention No. 2292895 (publication date: 10.02.2007, IPC: A61 K35 / 28). According to the known method, bone marrow aspirate is obtained during iliac crest puncture, layered on a density gradient (1.077 g / l), centrifuged for 30 min at a speed of 400 d. Then, a layer of mononuclear cells is collected from the phase boundary. In this case, 100 ml of bone marrow aspirate is taken with 20 ml of heparinized physiological solution with a heparin concentration of 2500 U / ml. Before layering, the heparinized bone marrow aspirate is preliminarily left to settle for 30 minutes at a temperature of 18-22°C, then the upper 2 / 3 of the cell suspension depleted of erythrocytes is collected, after which this sample is diluted 2-fold with heparinized phosphate-buffered saline with a heparin concentration of 10 U / ml.Next, after layering and centrifugation, the cell layer collected from the phase boundary is washed with heparinized phosphate-buffered saline with a heparin concentration of 10 U / ml by centrifugation three times for 10 min at 400 g and a temperature of 18-22°C, after which the cellularity of the suspension is brought to the desired concentration.

[0007] The disadvantage of the known method is its complexity, due to the need for manual collection of bone marrow aspirate fractions obtained as a result of centrifugation sedimentation. The multi-stage nature of the known method inevitably leads to mixing of components at the interfaces at its various stages. This may adversely affect the therapeutic effectiveness of the obtained bone marrow concentrate.

[0008] A method for treating knee osteoarthritis is known, disclosed in Russian Federation Patent for Invention No. 2685687 (publication date: 22.04.2019, IPC: A61 M 31 / 00, A61 K 35 / 28, A61 P 19 / 02, A61 B 17 / 34, BOU 21 / 26, A61 M 1 / 34). In the known method, the bone marrow concentrate is filtered and centrifuged before administration, while bone marrow is aspirated from the internal condyle of the tibia, filtration is carried out using a filter for blood transfusions, and centrifugation is carried out once at a speed of 3000 to 4000 rpm.

[0009] The disadvantage of the known method is the separation of the bone marrow aspirate into two fractions - erythrocyte sediment and bone marrow concentrate plasma. In this case, there is no separation of platelet-rich plasma, which is characterized by the highest content of mesenchymal cells. This can lead to obtaining a preparation characterized by their low concentration. In addition, the bone marrow aspirate of the tibial condyle is characterized by a relatively low concentration of mesenchymal cells.

[0010] A known method for producing an autologous bone marrow cell concentrate for the treatment of inflammatory and degenerative diseases in traumatology and orthopedics is disclosed in Russian Federation patent for invention No. 2763250 (publication date: 12 / 28 / 2021, IPC: A61 B 17 / 56, A61 M 1 / 34, A61 K 35 / 28, A61 P 19 / 02, BOU 21 / 26) - prototype. The known method includes taking aspirate of bone marrow from the iliac crest, performing a surgical approach 4-5 cm posterior to the medial border of the iliac crest, passing a trocar through the approach, installed in the middle of the cortical area of ​​the iliac crest, taking aspirate and centrifuging it, upon completion of which the mesenchymal cells of the bone marrow are counted.In this case, the tip of the trocar is passed through the cortical plate until a 35-50 mm gap is felt, bone marrow aspirate is collected in a volume of 50 ml, the bone marrow aspirate is centrifuged at a speed of no more than 2400 rpm for at least 20 minutes, while the concentration of mesenchymal cells in the resulting autologous bone marrow concentrate is at least 0.01%.

[0011] The disadvantage of the known method is the production of an autologous bone marrow concentrate, characterized by a relatively low concentration of mesenchymal cells. Among other things, this has a negative effect on the therapeutic effectiveness of the resulting drug.

[0012] DISCLOSURE OF THE ESSENCE OF THE INVENTION

[0013] The technical problem underlying the present invention is to obtain a biological preparation based on bone marrow concentrate.

[0014] The technical result of the claimed invention consists in increasing the therapeutic effectiveness of a biological preparation obtained from bone marrow aspirate.

[0015] An additional technical result consists in increasing the concentration of mesenchymal stem cells in a biological preparation obtained from bone marrow aspirate.

[0016] The invention proposes a method for obtaining an autologous bone marrow cell concentrate, which includes collecting a bone marrow aspirate with subsequent centrifugation and isolating a fraction containing mesenchymal cells. Unlike the prototype, the bone marrow aspirate is collected from the posterior iliac spine using a syringe with a needle with a mandrel. After that, the collected aspirate is introduced into the working chamber of the centrifuge and centrifuged at a speed of 3,000 to 4,000 revolutions per minute, wherein during centrifugation the said aspirate is illuminated with light beams with different wavelengths, and the light beams that have passed through the aspirate are recorded on a light sensor located opposite the light sources.In this case, the fraction containing mesenchymal cells, which is represented by platelet-rich plasma, platelet-depleted plasma, and erythrocyte sediment are removed from the centrifuge working chamber in turn, depending on the wavelengths of the light beams registered on the light sensor. After this, the platelet-rich plasma is diluted with an excess of platelet-depleted plasma and the resulting liquid is filtered from foreign inclusions.

[0017] Additional advantages and essential features of the present invention can be presented in the following particular embodiments.

[0018] In particular, bone marrow aspirate is collected under local anesthesia. A 2% lidocaine solution is used as anesthesia.

[0019] In particular, bone marrow aspirate is collected in a volume of 30 to 180 ml.

[0020] In particular, bone marrow aspirate is collected sequentially from biopsy points marked on the posterior iliac spine in a quantity of 1 to 6.

[0021] In particular, a 20G-88mm puncture needle is used as a syringe needle.

[0022] In particular, platelet-depleted plasma is removed from the centrifuge working chamber if light beams with wavelengths of 470 nm and 940 nm are simultaneously registered on the light sensor.

[0023] In particular, platelet-rich plasma is removed from the centrifuge working chamber if the light sensor does not register a light beam with a wavelength of 470 nm.

[0024] In particular, the erythrocyte sediment is removed from the working chamber of the centrifuge if the light sensor does not register a light beam with a wavelength of 940 nm.

[0025] In particular, red blood cell-rich plasma is withdrawn into a syringe for injection into the patient.

[0026] In particular, platelet-depleted plasma and erythrocyte sediment are removed into reservoir bags for subsequent disposal.

[0027] IMPLEMENTATION OF THE INVENTION

[0028] Red bone marrow contains hematopoietic and stromal cells. Hematopoietic cells, due to their ability to unlimited proliferation and differentiation, ensure the maintenance and renewal of the pool of formed elements of the blood. Bone marrow stromal cells are represented by osteoblasts, adipocytes, endothelial cells of blood vessels, smooth muscle cells, pericytes, stromal fibroblasts, and create a microenvironment for stem hematopoietic cells.

[0029] Predecessors of all types of cells necessary for the formation of stroma and maintenance of effective hematopoiesis are mesenchymal (stromal) cells. Mesenchymal cells can be isolated from adipose tissue, bone marrow, peripheral blood, umbilical cord, dental pulp, synovial membrane, amniotic fluid. In bone marrow, mesenchymal cells are present in small quantities (0.001-0.01%), however, it is often used as their source. In particular, the highest concentration of mesenchymal cells in the human body is observed in the wing of the ilium, namely, in the posterior iliac spine, where the concentration of mesenchymal cells in bone marrow aspirate reaches 28 CFU / ml.

[0030] Currently, there are many areas of using MSCs in clinical practice, including oncohematology. Their use is possible in case of hypofunction of the hematopoietic stem cell (HSC) transplant. MSCs are able to accelerate the process of engraftment of the introduced HSCs and, accordingly, the restoration of hematopoiesis by creating an optimal stromal microenvironment. Due to their immunomodulatory properties, MSCs have also found application in the treatment of the graft-versus-host reaction after transplantation of allogeneic HSCs.

[0031] The primary function of mesenchymal cells is to secrete growth factors and other cytokines (regulation of intercellular and intersystemic interactions in the body) to stimulate the process of proliferation (cell division and growth) and angiogenesis (formation of new blood vessels).

[0032] The maximum efficiency of mesenchymal cells occurs under the condition of their isolated high concentration. To achieve their high concentration, it is necessary to achieve the isolation of the corresponding fraction of bone marrow aspirate by removing fractions from the biological preparation, including thrombocytes, erythrocytes, nuclear cells, hematopoietic stem cells.

[0033] The proposed method for obtaining an autologous bone marrow cell concentrate is carried out as follows. Bone marrow aspirate is collected in an operating room under aseptic and antiseptic conditions. The patient is placed in a prone position. Before collecting the bone marrow aspirate, the collection site is disinfected, the skin and underlying soft tissues are locally anesthetized until the needle contacts the cortical plate. A 2% lidocaine solution in an amount of 2 ml is used as an anesthetic. After the anesthesia is administered, wait 1-2 minutes until the maximum analgesic effect is achieved. Aspirate is collected with a 20G-88MM puncture needle with a stylet from biopsy points marked in an amount of 1 to 6 on the skin area covering the posterior iliac spine. The needle is immersed through the cortical plate of the bone until a 35-50 mm gap is felt.The use of a syringe with a needle with a mandrel allows to exclude the ingress of solid fraction of bone crumbs, which enters the bone marrow aspirate when the bone is perforated with a trocar. The bone marrow aspirate is slowly drawn into the syringe. From each designated biopsy point, 30 ml of liquid enters the syringe. After a sufficient amount of bone marrow aspirate is in the syringe, the needle is removed and a bandage is applied to the skin surface.

[0034] To isolate the fraction enriched with mesenchymal cells, centrifugation is used, during which blood and bone marrow cells are separated in a density gradient due to differences in the buoyant density of blood cells. During centrifugation, blood cells move in the working chamber of the centrifuge until they reach the gradient region where their buoyant density is equal to the density of the medium. In this case, the buoyant density of platelet-depleted plasma is lower, so these cells are located in the upper layers of the centrifugate. The erythrocyte sediment, including granulocytes and erythrocytes, has a higher buoyant density and during centrifugation they sink to the bottom of the working chamber of the centrifuge.

[0035] Centrifugation of the entire volume of the taken bone marrow aspirate is carried out once at a speed of 3000-4000 rpm. Centrifugation at a speed of less than 3000 rpm does not allow separating the erythrocyte sediment from the plasma. Centrifugation at a speed of more than 4000 rpm leads to hemolysis of mesenchymal cells, as a result of which the therapeutic efficacy of the resulting biological preparation decreases. The centrifugation time depends on the volume of the aspirate introduced into the working chamber. The optimal centrifugation time for the introduced volume of aspirate is ensured by using a light sensor installed at the outlet of the centrifuge working chamber with the ability to register light beams with certain wavelengths.The bone marrow components are removed one by one into their designated containers depending on whether they absorb certain light beams or not, and these components are removed until the entire volume of bone marrow aspirate introduced into the working chamber of the centrifuge is separated into components. This allows for a purer and more effectively isolated target fraction containing an increased concentration of mesenchymal cells, which cannot be achieved by traditional centrifugal mechanical separation, as a result of which the various components of the aspirate inevitably mix.

[0036] Centrifugation is performed in a closed system from the centrifuge working chamber connected by sealed tubes through a distribution valve with a syringe for collecting the target fraction and two reservoir bags for collecting the erythrocyte sediment and platelet-depleted plasma. The fractions, separated in the centrifuge one by one depending on their buoyant density, are fed through a sealed tube to the distribution valve. The isolated bone marrow components are illuminated by light beams with wavelengths of 470 nm and 940 nm on their way to the distribution valve. The first component removed from the centrifuge working chamber is platelet-depleted plasma. When the plasma is removed, a light sensor located opposite the light sources records the passage of both light beams through the liquid moving in the tube.Based on the sensor reading, the distribution valve switches the tube for outputting bone marrow components to a sealed tube leading to a reservoir bag for collecting platelet-poor plasma. The second component output from the centrifuge working chamber is platelet-rich plasma. When platelet-rich plasma is output through the tube, the light sensor does not register a light beam with a wavelength of 470 nm, since platelets tend to absorb light with this wavelength. In this case, the distribution valve switches the tube for outputting bone marrow components to a port to which a syringe is connected for collecting the target fraction. The last component output from the centrifuge working chamber is the erythrocyte sediment, which has the highest buoyant density.When the erythrocyte sediment is removed through the tube, the light sensor does not register a light beam with a wavelength of 940 nm, since erythrocytes have the property of absorbing light with a specified wavelength. In this case, the distribution valve switches the tube for removing bone marrow components to a reservoir bag for collecting the erythrocyte sediment. Depending on the volume of the bone marrow aspirate, centrifugation takes from 15 to 20 minutes.

[0037] The platelet-rich plasma obtained as a result of centrifugation is collected in a syringe and is a ready-made biological preparation for injection into the patient as prescribed based on his symptoms. In this case, dividing the bone marrow aspirate into three components allows obtaining a concentrate of mesenchymal cells suitable for dilution with excess platelet-poor plasma. Such dilution is necessary in order to increase the volume of the preparation and, as a consequence, the area of ​​its active interaction with the internal organs of the patient into which the preparation is administered. Depending on the volume of the concentrate, dilution is carried out in the following approximate ratios: 1:3, 1:4, 1:5. For example, when obtaining 4 ml of a fraction containing mesenchymal cells, platelet-poor plasma is taken in a volume of 12 ml.As a result of dilution, the liquid is a fraction of bone marrow concentrate plasma with a concentration that is adjusted by the physician during the procedure for obtaining autologous bone marrow aspirate. The required final volume of the diluted preparation is determined by the physician depending on the injection site and indications for use. In turn, this increases the therapeutic efficacy of the preparation, as it makes it possible to obtain a preparation with a high concentration of mesenchymal cells in the volume required for administration. The diluted preparation is filtered on a filter with a pore diameter of no more than 3 μm under pressure or vacuum. As a result of filtration, the preparation is cleared of foreign inclusions, such as microscopic remains of bone chips or fat. The resulting filtrate is ready for reinfusion.Under microscopic magnification, the target fraction contains elongated, tightly adjacent spindle-shaped cells with a small number of processes.

[0038] A study of the obtained fraction of concentrated bone marrow showed that when centrifuging 40 ml of bone marrow aspirate containing mesenchymal cells in an amount of about 28 CFU / ml, the target fraction contains mesenchymal cells in an amount of about 843 CFU / ml, which indicates a concentration increase of approximately 30 times.

[0039] Comparison of the proposed method for obtaining an autologous bone marrow cell concentrate with the prototype shows the following. According to the prototype, centrifugation of the initial bone marrow aspirate containing about 0.001% mesenchymal cells leads to obtaining a concentrate containing about 0.01%, which indicates a concentration increase of approximately 10 times. Moreover, when diluting the bone marrow concentrate with depleted platelet mass, for example, in a ratio of 1:5, the concentration of mesenchymal cells in the preparation still exceeds the analogous indicator of the prototype by at least 2-3 times.

[0040] Thus, a comparison of the characteristics of the prototype and the proposed invention indicates a multiple increase in the concentration of mesenchymal stem cells in the biological preparation after processing the bone marrow aspirate, at least three times.

[0041] The proposed invention allows to increase the therapeutic effect of concentrated bone marrow, and as a result, find wide application in traumatology and orthopedics, plastic, aesthetic and burn surgery, etc.

Claims

CLAUSES OF THE INVENTION 1. A method for producing an autologous bone marrow cell concentrate, comprising collecting a bone marrow aspirate followed by centrifugation and isolating a fraction containing mesenchymal cells, characterized in that the bone marrow aspirate is collected from the posterior iliac spine using a syringe having a needle with a mandrel, said aspirate is introduced into the working chamber of the centrifuge and centrifuged at a speed of 3000 to 4000 revolutions per minute, and during centrifugation said aspirate is illuminated with light beams with wavelengths of 470 nm and 940 nm, and the light beams that have passed through the aspirate are recorded on a light sensor located opposite the light sources, while from the working chamber of the centrifuge a fraction containing mesenchymal cells, which is represented by platelet-rich plasma, platelet-poor plasma, and erythrocyte sediment, while platelet-rich plasma is removed from the working chamber of the centrifuge,if the light sensor does not register a light beam with a wavelength of 470 nm; platelet-depleted plasma is removed from the working chamber of the centrifuge if the light sensor simultaneously registers light beams with wavelengths of 470 nm and 940 nm; the erythrocyte sediment is removed from the working chamber of the centrifuge if the light sensor does not register a light beam with a wavelength of 940 nm; after which the platelet-rich plasma is diluted with excess platelet-depleted plasma and the resulting liquid is filtered from foreign inclusions.

2. The method according to paragraph 1, characterized in that the collection of bone marrow aspirate is carried out under local anesthesia.

3. The method according to paragraph 2, characterized in that a 2% lidocaine solution is used as anesthesia.

4. The method according to paragraph 1, characterized in that the bone marrow aspirate is collected in a volume of 30 to 180 ml.

5. The method according to paragraph 1, characterized in that the collection of bone marrow aspirate is performed sequentially from biopsy points marked on the posterior iliac crest in a quantity of 1 to 6.

6. The method according to paragraph 1, characterized in that a 20G-88mm puncture needle is used as the syringe needle.

7. The method according to claim 1, characterized in that the erythrocyte-rich plasma is withdrawn into a syringe for injection into the patient.

8. The method according to claim 1, characterized in that the unused platelet-poor plasma and erythrocyte sediment are withdrawn into reservoir bags for subsequent disposal.