JP5515133B2 - Bone regeneration composition manufacturing equipment - Google Patents

Description

  The present invention relates to a bone regenerating composition manufacturing device, a method for manufacturing a bone regenerating composition, a bone regenerating composition, and a bone regenerating method. In particular, the present invention is simple and quick in a closed system without performing a culture operation. In particular, the present invention relates to a bone regeneration composition production device capable of producing a bone regeneration composition, a method for producing a bone regeneration composition, a bone regeneration composition, and a bone regeneration method.

  As a method for repairing a bone defect or a bone union failure after a fracture, an autologous bone graft in which bone is collected from a healthy part other than the affected part of the patient and transplanted to the affected part is most commonly known. However, autologous bone transplantation has problems such as being invasive to the healthy part to be boned, the amount of bone being collected is limited, and there is a risk of complications such as fractures in the bone harvesting part. It was.

  Therefore, in recent years, a bone regeneration method has been developed in which a porous body mainly composed of calcium phosphate is filled into an affected area as a bone filler.

  However, it is considered that the ability to regenerate bone is insufficient only by filling with calcium phosphate, and its adaptation is limited to small defects with good regeneration conditions. Therefore, a new method has been developed in which mesenchymal stem cells are transplanted with calcium phosphate.

  In such a bone regeneration method, a bone regeneration composition containing mesenchymal stem cells and calcium phosphate is used. Such a bone regeneration composition and its manufacturing method are disclosed by patent documents 1-3, for example.

  Specifically, Patent Document 1 discloses a biocompatible, open-pore-type substrate in which the pores of the open-pore-type substrate are exposed to a vacuum, and the osteoinductive substance and / or the osteogenic substance is in a fluid form. A method for producing a bone substitute material containing osteoinductive material and / or osteogenic material by being sucked into these pores by a vacuum generated in the pores is disclosed.

  It is described that the open pore type substrate is at least partially made of a bioresorbable material, preferably hydroxyapatite or tricalcium phosphate.

  Further, the bone-forming substance is present in the form of a somatic cell in a fluid form, and the somatic cell is autologous bone marrow, isolated concentrated cell consisting of autologous bone marrow, cultured autologous stem cell, differentiated autologous stem cell, or It is described as a mesenchymal cell.

  In Patent Document 2, a desired cell composed of a hematopoietic stem cell, a mesenchymal stem cell, and a blood cell is pressed into a porous biological tissue filling material by pressure, and the desired cell is infiltrated into the biological tissue filling material. Thus, it is disclosed that a body tissue filling body to be filled in a body tissue defect part is manufactured.

  Specifically, a disk-shaped porous body made of β-tricalcium phosphate (hereinafter also referred to as “β-TCP”) is described as the biological tissue filling material.

  Patent Document 3 describes that a body tissue supplement is produced by separating a concentrate containing at least mesenchymal stem cells from a body fluid collected from a patient, and spraying the separated concentrate on the body tissue filling material. Has been.

  Specifically, a block-like or granular porous body made of β-TCP is described as the biological tissue filling material.

  Moreover, although it does not specialize in a bone regeneration composition or its manufacturing method, the technique as disclosed by patent documents 4 and 5 is known as a method of preparing stem cells, such as a mesenchymal stem cell.

  Specifically, Patent Document 4 discloses that tissue regeneration cells used for regeneration of living tissue excluding hematopoietic tissue and contaminated cells are passed through a cell separation filter, allowing the contaminated cells to pass through, and tissue regeneration. Disclosed is a method for separating cells for regeneration of biological tissue, which comprises a step of capturing fluid cells and then introducing a fluid into the cell separation filter and collecting the cells.

  Specifically, mesenchymal progenitor cells, vascular endothelial progenitor cells, and neural stem cells are described as the cells for regeneration of living tissue.

  Further, in Patent Document 4, cells for living tissue regeneration obtained by the above-described method for separating cells for living tissue regeneration are used as they are, or further separation and purification, culture, activation, amplification, gene transfer, cryopreservation as necessary. It can be used for treatment of lesions and / or defects in various living tissues and research in the basic science field after various treatments such as compounding with bone grafting materials such as hydroxyapatite and compounding with artificial blood vessels. It describes what you can do.

Patent Document 5 discloses that density K (that is, basis weight (g / m ² ) / thickness (m)) is 1.0 × 10 ⁴ ≦ K ≦ 1.0 × 10 ⁶ and the fiber diameter. Discloses a method for separating and recovering stem cells from a body fluid or a treatment solution of a biological tissue using a stem cell separation material of 3 to 40 μm or a stem cell separation filter formed by filling the stem cell separation material in a container.

  Specific examples of the stem cells include mesenchymal stem cells, pluripotent adult stem cells, and bone marrow stromal cells. Further, as the body fluid, specifically, bone marrow fluid, peripheral blood, or umbilical cord blood is described.

In addition to Patent Documents 4 and 5, as methods for preparing mesenchymal stem cells, 1) a method of separating from bone marrow fluid through a culture step, 2) a method of separating from bone marrow fluid using a cell separating agent, etc. Is also used.
Special table 2004-505747 gazette (announced February 26, 2004) JP 2003-320013 A (published on November 11, 2003) JP 2003-320019 A (published on November 11, 2003) Japanese Patent Laid-Open No. 2001-161352 (published on June 19, 2001) International Publication No. WO2007 / 046501 (Published April 26, 2007)

  As described above, a bone regeneration composition containing cells for regeneration of living tissue such as mesenchymal stem cells and a tissue repair material such as β-TCP is used for bone regeneration. Techniques for producing a regenerated composition are disclosed in Patent Documents 1 to 3.

  Since the above-mentioned bone regeneration composition is usually transplanted to a patient and used, it must be produced aseptically.

  However, in the techniques of Patent Documents 1 to 3, preparation (separation) of cells for regeneration of living tissue such as mesenchymal stem cells, and mixing of cells for regeneration of living tissue and a tissue filling material such as β-TCP Are performed as separate operations.

  Therefore, in the techniques of Patent Documents 1 to 3, there is a problem that a special device is required in order to mix the cells for living tissue regeneration and the tissue filling material while ensuring sterility. Moreover, since it is necessary to use such a special apparatus, there exists a problem that a bone regeneration composition cannot be manufactured simply.

  Furthermore, conventionally, for the production of a bone regenerating composition, cells for regenerating a living tissue separated from a body fluid or a treatment solution for living tissue are not used as they are, but after being separated and cultured once, the cultured living tissue It is common to use cells for regeneration.

  Therefore, a large cell processing center is required separately, and there is a problem that enormous costs such as capital investment and center maintenance costs are required.

  Thus, conventionally, a technique capable of easily producing a bone regeneration composition that can be used for clinical use has not been developed, and development of such a technique is eagerly desired.

  The present invention has been made in view of the above-mentioned problems, and the object thereof is a bone regeneration composition capable of easily and rapidly producing a bone regeneration composition in a closed system without performing a culture operation. It is providing the manufacturing instrument, the manufacturing method of a bone regeneration composition, the bone regeneration composition, and the bone regeneration method.

  As a result of intensive studies in view of the above problems, the present inventors have conducted separation of the bone regeneration-related cells from a sample containing bone regeneration-related cells involved in bone regeneration, and mixing of the bone regeneration-related cells and the support. The inventors have found that a bone regeneration composition can be easily and rapidly produced by a series of operations in a closed system, and have completed the present invention. That is, the present invention includes the following industrially useful inventions.

  (1) A bone regeneration composition-producing device for producing a bone regeneration composition comprising a bone regeneration-related cell involved in bone regeneration and a support that supports the bone regeneration-related cell, the bone regeneration-related cell A cell capture unit for capturing the cell, and a cell recovery solution injection unit for injecting a cell recovery solution into the cell capture unit and causing the bone regeneration-related cells captured by the cell capture unit to flow out of the cell capture unit And a cell recovery unit for recovering a cell recovery solution containing the bone regeneration-related cells that have flowed out of the cell trapping unit, wherein the cell recovery unit is provided with a support, and the cell recovery unit The bone regeneration composition-producing device, wherein the bone regeneration-related cells collected in the cell collection unit and the support are mixed.

  (2) The cell recovery unit can be detached from the bone regeneration composition manufacturing instrument, and the cell recovery unit includes a container having a protrusion, and a support enclosing unit enclosing the support. A cell collection liquid containing the bone regeneration-related cells by removing the cell collection part from which the cell collection liquid containing the bone regeneration-related cells is collected. When the centrifugation is performed, the bone regeneration-related cell precipitate can be stored, and the connection portion opens and closes the bone regeneration-related cell from the container portion to the support storage portion. The device for producing a bone regeneration composition according to (1), which is for controlling the movement of the precipitate.

  (3) The bone regenerating composition producing device according to (2), wherein the support enclosing portion is removable from the cell recovery portion.

(4) The bone regenerating composition producing device according to (2) or (3), wherein the support enclosing portion is made of a pressurizable material.
(5) The cell recovery unit includes a first space and a second space separated by a filtration unit, and the support is disposed in the first space, and the filtration unit includes the first space. (1) The cell recovery solution containing the bone regeneration-related cells recovered in the space is filtered, and components other than the bone regeneration-related cells are allowed to flow into the second space. Bone regeneration composition manufacturing equipment.

  (6) A sample injection unit for injecting a sample containing the bone regeneration-related cells into the cell capture unit is further provided, and the sample injection unit is configured such that the cell recovery solution injection unit The bone regenerative composition production instrument according to any one of (1) to (5), wherein the sample is injected into the cell trapping part in a direction opposite to the direction in which the recovery liquid is injected.

  (7) When the sample injection unit injects the sample into the cell capture unit, the sample injection unit further includes a waste liquid recovery unit for recovering a component of the sample that has passed through the cell capture unit ( The bone regeneration composition manufacturing apparatus as described in 6).

  (8) The apparatus further includes a washing liquid injecting unit for injecting a washing liquid for washing the cell capturing unit into the cell capturing unit, and the washing liquid injecting unit is configured so that the cell recovery liquid injecting unit is in contact with the cell capturing unit. The bone regenerating composition producing device according to any one of (1) to (7), wherein the washing liquid is injected into the cell trapping part in a direction opposite to the direction in which the cell recovery liquid is injected. .

  (9) The bone regenerating composition producing device according to any one of (1) to (8), wherein the cell trapping part includes a cell separation material having a filter structure.

  (10) The cell separating material is characterized in that when a sample containing leukocytes and erythrocytes is passed, 30% to 90% of leukocytes and 90% or more of erythrocytes are allowed to pass (9). The bone regeneration composition manufacturing instrument described in 1.

  (11) The bone regeneration composition-producing apparatus according to any one of (1) to (10), wherein the bone regeneration-related cell is a mesenchymal stem cell.

  (12) The support includes calcium phosphate, a natural polymer material, a synthetic polymer material, a metal material, or a composite material obtained by combining two or more of these materials. The bone regeneration composition manufacturing instrument according to claim 1.

  (13) A method for producing a bone regeneration composition comprising a bone regeneration-related cell involved in bone regeneration and a support that supports the bone regeneration-related cell, wherein the cell regeneration unit captures the bone regeneration-related cell in a closed system. The bone regeneration-related cells captured by the cell capture unit are caused to flow out of the cell capture unit by injecting the cell recovery solution into the cell capture unit, and the bone regeneration that has flowed out of the cell capture unit Bone regeneration characterized by collecting a cell recovery solution containing related cells in a cell recovery unit, concentrating the bone regeneration-related cells in the cell recovery unit, and then mixing the bone regeneration-related cells and a support. A method for producing the composition.

  (14) Before causing the bone regeneration-related cells to flow out from the cell capture unit using the cell recovery solution, a washing solution is applied in a direction opposite to the direction in which the cell recovery solution is injected into the cell capture unit. The method for producing a bone regeneration composition according to (13), wherein the cell capturing part is washed.

  (15) The method for producing a bone regeneration composition according to (13) or (14), wherein the bone regeneration-related cells collected in the cell collection unit are mixed with the support without culturing.

  (16) The method for producing a bone regeneration composition according to any one of (13) to (15), wherein the bone regeneration-related cell is a mesenchymal stem cell.

  (17) Any of (13) to (16), wherein the support includes calcium phosphate, a natural polymer material, a synthetic polymer material, a metal material, or a composite material obtained by combining two or more of these. A method for producing the bone regeneration composition according to claim 1.

  (18) A bone regeneration composition manufactured using the bone regeneration composition-producing device according to any one of (1) to (12).

  (19) A bone regeneration composition produced using the method for producing a bone regeneration composition according to any one of (13) to (17).

(20) The bone regeneration composition according to (18) or (19), wherein 1 to 5 × 10 ⁹ bone regeneration-related cells are contained per 100 mg of the support.

  (21) A bone regeneration method comprising regenerating bone using the bone regeneration composition according to any one of (18) to (20).

  As described above, the bone regenerating composition manufacturing device according to the present invention includes a cell capturing unit for capturing bone regeneration-related cells involved in bone regeneration, and injecting a cell recovery solution into the cell capturing unit. A cell recovery solution injection unit for allowing the bone regeneration-related cells captured by the cell to flow out of the cell capture unit, and a cell for recovering a cell recovery solution containing the bone regeneration-related cells that have flowed out of the cell capture unit And a recovery unit. Further, a support is disposed in the cell recovery unit, and the bone regeneration-related cells recovered in the cell recovery unit and the support are mixed in the cell recovery unit.

  Therefore, there is an effect that a bone regeneration composition including a bone regeneration-related cell and a support that supports the bone regeneration-related cell can be easily produced in a closed system.

  An embodiment of the present invention will be described below with reference to FIGS. 1 to 3, but the present invention is not limited to this.

<I. Bone Regeneration Composition Manufacturing Equipment>
The bone regeneration composition producing device according to the present invention includes (1) separating bone regeneration-related cells from body fluids, and (2) separating the bone regeneration-related cells and mixing them with the bone regeneration-related cells. It is possible to mix with a support that can be used as a series of operations in a closed system.

  Therefore, according to the bone regeneration composition manufacturing apparatus concerning this invention, a bone regeneration composition can be manufactured simply.

  Moreover, according to the bone regeneration composition manufacturing apparatus concerning this invention, it is not necessary to culture | cultivate a bone regeneration related cell in the manufacturing process of a bone regeneration composition. Therefore, it is possible to greatly reduce the labor and manufacturing cost for manufacturing the bone regeneration composition.

  In the present specification, the “bone regeneration composition” includes at least a bone regeneration-related cell and a support, and is intended for a composition for use in bone regeneration.

  In the present specification, “bone regeneration-related cells” are intended to be cells involved in bone regeneration, and specific examples include mesenchymal stem cells.

  In the present specification, the term “mesenchymal stem cell” is intended to be a cell that is isolated from a body fluid, has the ability to repeat self-proliferation, and can be differentiated into a downstream lineage. Specifically, bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells and the like can be mentioned. These mesenchymal stem cells have the ability to differentiate not only into cells of their origin lineage but also into cells of other lineages. Here, the property of mesenchymal stem cells will be described.

  Mesenchymal stem cells are differentiated depending on differentiation-inducing factors, such as chondrocytes, vascular endothelial cells, cardiomyocytes, adipose tissue, or cement blasts or periodontal ligament fibroblasts that constitute periodontal tissues. Differentiates into germ layer cells.

  In addition, mesenchymal stem cells are known to differentiate into osteoblasts and to regenerate bone tissue under an appropriate environment. In this bone tissue neoplasia, mesenchymal stem cells are thought to play a role to compensate for the lack of osteogenic ability.

  In the present specification, the “support” is intended to be a support that can be mixed with bone regeneration-related cells and used as a bone regeneration composition. That is, it supports bone regeneration-related cells.

  The support is not particularly limited. Specifically, for example, calcium phosphate such as hydroxyapatite, β-tricalcium phosphate, calcium carbonate, or octacalcium phosphate; collagen, chitosan, chitin, etc. Natural polymer materials; synthetic polymer materials such as polylysine, polyglycolic acid, polylactic acid, polycyanoacrylate, or ε-caprolactan; metal materials such as pure titanium, titanium alloys, tantalum, or stainless steel it can.

  These may be used singly or as a composite material combining two or more.

  In addition, tissues such as autologous bones, autologous bone fragments, autologous bone meal, and bone fragments with vascular handles may be used as the support.

  The shape of the support is not particularly limited as long as it can hold bone regeneration-related cells. Specifically, for example, a granular shape, a membrane shape, a tube shape, a flat plate shape, a roll shape and the like can be mentioned. Especially, in order to make a shape fit a diseased part, a granular form is preferable.

  Here, although one embodiment of the bone regeneration composition manufacturing instrument concerning this invention is described in detail below, referring FIGS. 1-3, this invention is not limited to this.

  As shown in FIG. 1, a bone regenerating composition manufacturing device 100 (bone regenerating composition manufacturing device) according to this embodiment includes a cell separator 1 (cell trapping unit) and a sample injection syringe 2 (sample injection unit). And a waste liquid bag 3 (waste liquid recovery section), a cleaning syringe 4 (cleaning liquid injection section), a recovery syringe 5 (cell recovery liquid injection section), and a mixing container 6 (cell recovery section).

  The cell separator 1, the waste liquid bag 3, and the collection syringe 5 are connected to each other via a three-way cock 12.

  More specifically, as shown in FIG. 1, the cell separator 1 and the three-way stopcock 12 are connected by a piping circuit 18. The waste liquid bag 3 and the three-way cock 12 are connected by a piping circuit 20.

  Further, the collection syringe 5 and the three-way stopcock 12 are connected by a piping circuit 19. Thereby, as a result, the cell separator 1, the waste liquid bag 3, and the collection syringe 5 are connected to each other.

  The cell separator 1, the sample injection syringe 2, the washing syringe 4, and the mixing container 6 are connected to each other via a three-way stopcock 10 and a three-way stopcock 11.

  More specifically, as shown in FIG. 1, the cell separator 1 and the three-way stopcock 11 are connected by a piping circuit 17. The mixing container 6 and the three-way cock 11 are connected by a piping circuit 16. The three-way stopcock 11 and the three-way stopcock 10 are connected by a piping circuit 15.

  Further, the cleaning syringe 4 and the three-way cock 10 are connected by a piping circuit 14. Furthermore, the sample injection syringe 2 and the three-way cock 10 are connected by a piping circuit 13.

  Thereby, as a result, the cell separator 1, the sample injection syringe 2, the washing syringe 4, and the mixing container 6 are connected to each other.

  According to the above configuration, when a sample solution containing bone regeneration-related cells is injected from the sample injection syringe 2, the sample solution can be flowed into the cell separator 1 via the three-way stopcock 10 and the three-way stopcock 11. .

  In the cell separator 1, bone regeneration-related cells contained in the sample solution are captured by the cell separator 1, and impurities contained in the sample solution pass through the three-way stopcock 12 and are disposed in a waste solution bag. 3 can flow into (discharge).

  Moreover, according to the said structure, when a washing | cleaning liquid is inject | poured from the syringe 4 for washing | cleaning in the state by which the bone regeneration related cell was capture | acquired by the cell separator 1, it will pass through the three-way cock 10 and the three-way cock 11 The cleaning liquid can be caused to flow into 1.

  As a result, the washing liquid flows into the cell separator 1 in the same direction as when the sample solution flows. Therefore, the contaminants in the cell separator 1 can be washed away, and the washing solution can flow (discharge) into the waste solution bag 3 via the three-way stopcock 12.

  Furthermore, according to the above configuration, when the cell recovery liquid is injected from the recovery syringe 5 in a state where the bone regeneration-related cells are captured by the cell separator 1 and the contaminants in the cell separator 1 are washed away, the three-way stopcock The cell recovery solution can be allowed to flow into the cell separator 1 via 12.

  As a result, the cell recovery solution flows into the cell separator 1 in the opposite direction to that when the sample solution is introduced. Therefore, the bone regeneration-related cells captured by the cell separator 1 can flow out of the cell separator 1, and the bone regeneration-related cells can flow into the mixing container 6 via the three-way cock 11.

  As shown in FIG. 1, a support 7 is disposed in the mixing container 6 of the bone regenerating composition manufacturing device 100. Therefore, the bone regeneration-related cells that have flowed into the mixing container 6 are mixed with the support 7 in the mixing container 6.

  As the support 7, the above-described support may be used. Moreover, since the method for mixing the bone regeneration-related cells and the support 7 in the mixing container 6 will be described later, the detailed description thereof is omitted here.

  Thus, according to the bone regeneration composition manufacturing instrument 100, the bone regeneration-related cells are separated from the sample solution containing the bone regeneration-related cells, and further, the separated bone regeneration-related cells and the support 7 are mixed. Can be performed as a series of operations in a closed system. Therefore, a bone regeneration composition can be easily produced while maintaining sterility.

  Furthermore, the bone regeneration composition produced by the bone regeneration composition production device 100 is not capable of culturing bone regeneration-related cells in the production process. Have That is, according to the bone regeneration composition manufacturing device 100, a bone regeneration composition having bone regeneration ability can be manufactured without culturing bone regeneration-related cells. Therefore, the step of culturing bone regeneration-related cells can be omitted, and the production efficiency of the bone regeneration composition can be greatly improved.

  Hereinafter, each member which comprises the bone regeneration composition manufacturing instrument 100 is demonstrated more concretely.

  The cell separator 1 can capture bone regeneration-related cells and allow unnecessary impurities to pass therethrough. As used herein, “contaminants” are intended to mean cells other than target cells, such as red blood cells, platelets, lymphocytes, and the like.

  The form of the cell separator 1 is not particularly limited, and a cell separator having an arbitrary size and shape may be filled in a container having at least two ports, or may be filled in a container or the like. The cell separation material itself may not be used.

  In the present embodiment, the cell separator 1 is preferably one in which a cell separation material having a filter structure is filled in a container. In this case, the cell separation material may be compressed and filled in the container, or may be filled without being compressed.

  More specifically, for example, a non-woven cell separator is filled in a thickness of 0.1 cm to 5 cm, preferably 0.15 cm to 4 cm, more preferably 0.2 cm to 3 cm. What filled the said container can be used suitably as the cell separator 1. FIG.

  According to the cell separator 1 having such a configuration, bone regeneration-related cells can be recovered at a high recovery rate while efficiently removing red blood cells, white blood cells, platelets, and the like.

  Further, when a roll-shaped cell separation material is used as the cell separation material, the roll-shaped cell separation material is filled in the container, and a body fluid is flowed from the inside to the outside of the roll so that bone regeneration-related cells are obtained. It is good also as a form to capture, It is good also as a form which flows a bodily fluid toward the inner side from the outer side of a roll, and capture | acquires a bone regeneration related cell.

  The container filled with the cell separation material is not particularly limited, and may take any form such as a sphere, a container, a cassette, a bag, a tube, and a column. Preferable specific examples include, for example, a transparent or translucent cylindrical container having a capacity of about 0.1 ml to 1000 ml and a diameter of about 0.1 cm to 15 cm, or a square or rectangle having a length of about 0.1 cm to 20 cm. And a square columnar container having a thickness of about 0.1 cm to 5 cm.

  The said cell separation material should just be a filtration material which allows a liquid component to pass through without a support body and a bone regeneration related cell passing. Specifically, when a sample containing leukocytes and erythrocytes is passed, it is preferable to pass 30% to 90% of leukocytes and more than 90% of erythrocytes. The above-mentioned “sample containing leukocytes and erythrocytes” includes bone marrow fluid, peripheral blood, a product obtained by treating a body fluid with a separating agent using a density gradient (a body fluid treated with a separating agent is an arbitrary liquid (medium, physiological Samples resuspended in saline, PBS, etc.) are intended as the separating agent, such as Ficoll, hydroxyethyl starch and the like.

  Examples of the material of the cell separation material include polyolefin such as polypropylene, polyethylene, high density polyethylene, and low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, poly Hydroxyethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass And cotton.

  Among these, synthetic polymers such as polyester, polystyrene, acrylic, rayon, polyolefin, vinylon, nylon, and polyurethane can be suitably used.

  The cell separation material may be composed of a single material among these materials, or may be composed of a composite material obtained by combining a plurality of materials.

  When two or more synthetic polymers are used in combination, the combination is not particularly limited, but a combination of polyester and polypropylene, a combination of rayon and polyolefin, or a combination of polyester, rayon and vinylon. It can be used suitably.

  When combining two or more kinds of synthetic polymers into fibers, specifically, for example, one fiber may be a fiber made of a synthetic polymer of different components, or may be a split fiber in which different components are separated from each other. . Moreover, the fiber which each compounded the fiber which consists of a synthetic polymer different in a component may be sufficient. As used herein, “complexed fiber” is intended to be a fiber formed by mixing two or more kinds of fibers, or a fiber in which fibers made of a synthetic polymer alone are bonded together.

  The form of the cell separator is not particularly limited, but in the present invention, it is preferable to use a cell separator 1 filled with a cell separator having a filter structure as the cell separator 1. It is preferable that the form of the material can be stored in the container.

  Specifically, for example, a porous body having a communication hole structure, an aggregate of fibers, a woven fabric, and the like can be given. Especially, it is preferable that it is comprised with a fiber and it is more preferable that it is a nonwoven fabric.

  In addition, the cell separation material may be a material obtained by performing a treatment for improving the performance as a cell separation material on the exemplified materials.

  Specifically, such treatment includes, for example, (1) hydrophilization treatment, (2) cell adhesion protein, and immobilization of antibodies against specific antigens specifically expressed on bone regeneration-related cells. Can be mentioned.

  By the hydrophilic treatment, non-specific capture of cells other than bone regeneration-related cells can be suppressed. Further, when the body fluid passes through the cell separator 1, it can be prevented that it passes biased toward the cell separation material in a specific region and the recovery rate of the bone regeneration-related cells is reduced.

  In addition, according to the above-described specific protein immobilization treatment, the adhesion of bone regeneration-related cells to the cell separation material can be further improved.

  The specific method for the hydrophilic treatment and the specific protein immobilization treatment is not particularly limited, and a conventionally known method may be used. Moreover, about the process which improves the performance of a cell separation material, the content of the said patent document 5 is also used for reference in this specification, for example.

  Since the cell separator 1 is used for separating bone regeneration-related cells, it is preferable that the cell separating material has a form in which holes are communicated in parallel with the liquid passing direction.

  In addition, when the cell separation material is a fiber, if the fiber diameter is smaller than 3 μm, the interaction between the cell separation material and the white blood cell tends to increase, and the removal efficiency of impurities (in other words, unnecessary cells) tends to decrease. is there. On the other hand, if the fiber diameter is larger than 40 μm, the effective contact area tends to be reduced and a short pass tends to occur, and the recovery rate of bone regeneration-related cells tends to decrease.

  Therefore, when the cell separation material is a fiber, the fiber diameter is preferably 3 μm to 40 μm, more preferably 5 μm to 35 μm, and still more preferably 5 μm to 30 μm. With such a fiber diameter, the interaction between the bone regeneration-related cells and the cell separation material can be increased, and the yield of the bone regeneration-related cells can be improved.

  Furthermore, when the cell separation material is a fiber, if the mesh opening is smaller than 3 μm, the removal efficiency of impurities tends to decrease. On the other hand, when the mesh opening is larger than 120 μm, it tends to be difficult to capture bone regeneration-related cells.

  Therefore, the mesh has a minor axis of 3 μm or more and a major axis of preferably 120 μm or less, a minor axis of 5 μm or more, and a major axis of 80 μm or less, a minor axis of 5 μm or more, and a major axis. Is more preferably 50 μm or less. With such an opening, relatively large contaminants such as red blood cells can be removed with high removal efficiency, and bone regeneration-related cells can be captured at a high recovery rate.

  The “fiber opening when the cell separation material is a fiber” here can be determined by the following method. First, the fiber in the case where the cell separation material is a fiber is photographed with a scanning electron microscope (hereinafter also referred to as “SEM”). Next, using the SEM image, the major and minor diameters of the substantial holes formed by the crossing of two or more different fibers are measured by an image analyzer at 50 points or more, and the average value of each is obtained. Ask for. Thereby, the minor axis and major axis of the mesh can be measured, respectively.

  The major axis refers to the longest distance between two points of the substantial hole, and the minor axis refers to the shortest distance substantially passing through the median of the distance between the two points when the major axis is obtained.

In the cell separator 1, the density K (that is, the basis weight (g / m ² ) / thickness (m)) of the cell separator is preferably 1.0 × 10 ⁴ ≦ K ≦ 1.0 × 10 ^6. 2.5 × 10 ⁴ ≦ K ≦ 7.5 × 10 ⁵ , more preferably 5.0 × 10 ⁴ ≦ K ≦ 5.0 × 10 ⁵ , and 5.0 × 10 ^5. It is particularly preferable that ⁴ ≦ K ≦ 2.0 × 10 ⁵ . With such a density, red blood cells, white blood cells, platelets and the like can be removed with high removal efficiency, and bone regeneration-related cells can be collected with a high recovery rate.

The density K indicates the basis weight (g / m ² ) / thickness (m). In other words, the density K can also be expressed as weight (g) / unit volume (m ³ ). Therefore, the density K can be determined by measuring the weight (g) per unit volume (m ³ ) of the cell separation material.

  In addition, the cell separator 1 can also be configured to include only the cell separator without including the container. In that case, specifically, a filter, a porous body, a hollow fiber, a mesh, and the like obtained by molding the cell separation material can be exemplified.

  Although the cell separator 1 is as having demonstrated above, about the structure of the cell separator 1, the content of the said patent document 5 is also used as reference in this specification, for example.

  The sample injection syringe 2 is for injecting a sample solution containing bone regeneration-related cells (hereinafter also referred to as “bone regeneration-related cell-containing sample”) into the cell separator 1.

  The material and shape of the sample injection syringe 2 are not particularly limited, and any conventionally known syringe, preferably a medical syringe can be used.

  In the bone regeneration composition manufacturing instrument 100 according to this embodiment, the sample regeneration syringe 2 is provided to inject the bone regeneration-related cell-containing sample into the cell separator 1, but the present invention is not limited to this. That is, instead of the sample injection syringe 2, any sample injection tool can be used depending on the method of injecting the bone regeneration-related cell-containing sample into the cell separator 1.

  Specifically, for example, instead of the sample injection syringe 2, an arbitrary container (for example, a bag) is used, and the bone regeneration-related cell-containing sample is pooled in the arbitrary container, and the cell separator is separated by natural fall. 1 can be injected with a sample containing bone regeneration-related cells. Alternatively, the bone regeneration-related cell-containing sample may be injected into the cell separator 1 from any container containing the bone regeneration-related cell-containing sample using a pump or the like.

  The washing syringe 4 and the collection syringe 5 are for injecting the washing solution and the cell collection solution into the cell separator 1, respectively. The material and shape of the washing syringe 4 and the collection syringe 5 are not particularly limited, but the same material as the sample injection syringe 2 can be used. Further, in the same manner as the sample injection syringe 2, a substance other than the syringe can be used depending on the method of injecting the washing liquid or the cell recovery liquid into the cell separator 1.

  The waste liquid bag 3 collects the components that have passed through the cell separator 1 out of the bone regeneration-related cell-containing sample and the cleaning liquid injected from the sample injection syringe 2 and the cleaning syringe 4. The material and shape of the waste liquid bag 3 are not particularly limited, and a waste liquid bag generally used for medical use can be suitably used.

  Moreover, although the waste liquid bag 3 is provided in this embodiment, this invention is not limited to this. That is, the bone regeneration-related cell-containing sample and the cleaning solution injected from the sample injection syringe 2 and the cleaning syringe 4 are not limited to bags as long as the components that have passed through the cell separator 1 can be recovered. Any container can be substituted.

  Each of the three-way stopcocks 10 to 12 has three ports, and any material can be used as long as it can communicate any two of these three ports. The material and the like are not particularly limited. Specifically, for example, any three-way stopcock used for medical purposes can be used.

  Furthermore, the piping circuits 13 to 20 are not particularly limited as long as the members can be connected so that the solution does not leak. The material and shape are not particularly limited. Specifically, for example, all tubes used in medical applications can be suitably used.

  Hereinafter, the mixing container 6 will be described in detail. Although three embodiments of the mixing container 6 are described here, the present invention is not limited to this.

[First Embodiment of Mixing Container 6]
As shown in FIG. 1, the mixing container 6 has a configuration in which a filter 8 (filtration unit) is disposed in a container 21. More specifically, the filter 8 is disposed so as to bisect the internal space of the container 21. As a result, the mixing container 6 has a mixing portion 9a (first space) and a storage portion 9b (second space) separated by the filter 8.

  In the mixing container 6, the mixing unit 9 a is a space through which the solution flowing into the mixing container 6 first passes. On the other hand, the reservoir 9b is a space in which the solution flowing into the mixing container 6 is stored after passing through the filter 8.

  The filter 8 is for filtering the cell collection liquid containing the bone regeneration-related cells collected (inflowed) into the mixing unit 9a and allowing components other than the bone regeneration-related cells to flow into the storage unit 9b. The filter 8 is not particularly limited, but is preferably a filter that substantially does not pass particles as large as cells and allows only components having a particle size smaller than cells to pass through. Specifically, for example, it is preferable to use a filter having a communicating hole that is parallel to the liquid flow direction and having a hole diameter of 2 μm to 5 μm.

  Furthermore, in the mixing container 6, the support body 7 is arrange | positioned in the space (for example, on the filter 8) of the mixing part 9a. According to such a configuration, the solution flowing into the mixing unit 9a of the mixing container 6 is mixed with the support 7 in the mixing unit 9a.

  Therefore, according to the above configuration, the cell recovery solution containing the bone regeneration-related cells recovered in the mixing container 6 (in other words, the cell suspension of the bone regeneration-related cells) is mixed with the support 7 in the mixing unit 9a. After that, the components excluding the bone regeneration-related cells are moved to the reservoir 9b. As a result, a mixture of bone regeneration-related cells and the support remains on the filter 8.

  And by inject | pouring arbitrary solutions into the mixing part 9a, the bone regeneration composition containing the concentrated bone regeneration related cell and a support body can be manufactured.

  A method for injecting an arbitrary solution into the mixing unit 9a and a method for recovering the manufactured bone regeneration composition from the mixing unit 9a are not particularly limited. Specifically, for example, using a syringe or the like. An arbitrary solution can be injected into the mixing unit 9a, or the bone regeneration composition can be recovered from the mixing unit 9a.

  The container 21 is not particularly limited, and any container can be used. The container 21 may be a hard container or a soft container. Specific examples of the material of the container 21 include polyolefins such as polypropylene, polyethylene, high density polyethylene, and low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, vinylon, polystyrene, acrylic (polymethyl methacrylate, (Polyhydroxyethyl methacrylate, acrylonitrile, acrylic acid, acrylate ester, etc.), nylon, polyurethane, polyimide, polyamide, carbon, polyacrylate, cellulose, chitin, chitosan, and glass.

  The container 21 may be made of a single material, or may be a combination of a plurality of materials as exemplified above.

[Second Embodiment of Mixing Container 6]
As shown in FIG. 2, the mixing container 6 includes a container 26 (container part) having a protrusion 27, a support enclosing container 29 (support enclosing part) in which the support 7 is encapsulated, as shown in FIG. 2. And the tube 28 (connecting portion).

  The protrusion 27 of the container 26 and the support enclosing container 29 are connected by a tube 28. Further, the container 26 and the support enclosing container 29 are in a non-communication state when the tube 28 is closed. On the other hand, when the tube 28 is in the open state, the container 26 and the support-enclosed container 29 are in communication.

  That is, the tube 28 is for controlling the movement of the solution from the container 26 to the support enclosing container 29. The tube 28 may be reversibly openable and closable, or may be switchable only once from the closed state to the open state.

  Examples of the form that can be switched only once from the closed state to the open state include a form in which the tube 28 is a non-communicatable tube that can be opened, and is opened by bending the tube. The opening / closing method of the tube 28 is not limited to the above, and any opening / closing method can be used.

  In the bone regeneration composition manufacturing instrument 100 including the mixing container 6 of the present embodiment, the mixing container 6 is preferably removable from the bone regeneration composition manufacturing instrument 100.

  According to such a configuration, after recovering a cell recovery solution containing bone regeneration-related cells (in other words, a cell suspension of bone regeneration-related cells) in the mixing container 6, the bone regeneration composition producing device 100 is used. Can be removed. Furthermore, the removed mixing container 6 can be subjected to a centrifuge to concentrate bone regeneration-related cells and settle (contain) in the protrusion 27.

  That is, when the protrusion 27 removes the mixing container 6 that collects the cell recovery solution containing bone regeneration-related cells and centrifuges the cell recovery solution containing bone regeneration-related cells, the protrusion 27 precipitates the bone regeneration-related cells. Can be stored.

  Thereafter, when the tube 28 is opened, the protrusion 27 and the support enclosing container 29 communicate with each other, and the concentrated bone regeneration-related cells flow into the support enclosing container 29, and the mesenchymal cells and the support 7 can be mixed to prepare (manufacture) a bone regeneration composition.

  The material and shape of the container 26 are not particularly limited, but the material can withstand the operation of centrifuging the suspension of bone regeneration-related cells collected in the container 26 to precipitate the bone regeneration-related cells. And the shape is preferred.

  Specifically, for example, the material of the centrifuge tube or the centrifuge bag that is generally used to precipitate cells by centrifugation can be selected and suitably used from those exemplified as the material of the container 21. .

  Further, regarding the shape of the container 26, specifically, the bone regeneration-related cell suspension collected in the container 26 can be centrifuged to precipitate the bone regeneration-related cell. Any shape that can accommodate the related cells in the protrusion 27 may be used.

  The material and shape of the support enclosure 29 are not particularly limited as long as the support 7 can be enclosed. Specifically, for example, a container made of the material exemplified as the material of the container 21 can be used.

  In the bone regenerating composition producing device 100, the support-enclosed container 29 is preferably in a negative pressure state, preferably a vacuum, before communicating with the container 26, that is, when the tube 28 is closed.

  According to such a configuration, when the support enclosing container 29 is communicated with the container 26, more than necessary detail recovery liquid does not flow into the support enclosing container 29 and settles on the protrusion 27. Bone regeneration-related cells (in other words, concentrated bone regeneration-related cells) preferentially flow into the support enclosure 29. Therefore, a bone regeneration composition having a high cell concentration of bone regeneration-related cells can be produced.

  The support enclosing container 29 is preferably a pressurizable container. The container capable of pressurization is intended to be a container capable of reducing the space of the container by applying external pressure. Further, the support enclosing container 29 is preferably separable from the container 26.

  According to the above configuration, after the bone regeneration composition is prepared in the support enclosing container 29, the support enclosing container 29 is separated from the container 26 together with the tube 28, or only the support enclosing container 29 is separated from the container 26. By applying an external pressure to the container 29, the bone regeneration composition can be pushed out and collected through the tube 28 or the opening of the support enclosing container 29.

  Further, by applying external pressure to the support enclosing container 29, the bone regeneration composition can be directly transplanted to the diseased part via the tube.

  More specifically, for example, if a syringe is used as the support enclosing container 29, an external pressure is applied to the support enclosing container 29 by pushing a piston, and the bone regeneration composition is recovered from the support enclosing container 29. Can be transplanted directly to the diseased part.

  If a soft container is used as the support enclosing container 29, external pressure is applied to the support enclosing container 29 by pushing the support enclosing container 29 with a finger or the like, and the bone regeneration composition is recovered from the support enclosing container 29. Or transplanted directly to the affected area.

  Furthermore, if a hard container is used as the support enclosing container 29, an external pressure is applied to the support enclosing container 29 using a machine such as a pump, and the bone regeneration composition can be recovered from the support enclosing container 29 or used in a diseased part. Or can be transplanted directly.

  The method for recovering the bone regeneration composition from the support-enclosed container 29 is not limited to the method using pressurization. For example, the bone regeneration composition may be recovered from the support enclosure 29 using a syringe or the like.

[Third Embodiment of Mixing Container 6]
As shown in FIG. 3A, the mixing container 6 can be connected to a container 47 (container part) having a protrusion part 48 (protrusion part) and a protrusion part 48 of the container 26, as shown in FIG. It consists of the support body enclosure 50 (support body enclosure part) in which the support body 7 was enclosed.

  In this embodiment, the container 47 and the support inclusion body 50 are not in communication until the cell collection liquid containing the bone regeneration-related cells (in other words, the cell suspension of the bone regeneration-related cells) is collected in the container 47. Is in a state. Specifically, in FIG. 3A, the container 47 and the support enclosure 50 are in a separated state. However, the present invention is not limited to this, and the container 47 and the support enclosure 50 are connected without being completely separated, and the support enclosure 50 is pushed in the direction of the container 47. Thus, the needle 51 can be stuck in the protrusion 48 of the container 47 so that the container 47 and the support enclosure 50 communicate with each other.

  Further, the protrusion 48 of the container 47 is covered until the cell suspension of the bone regeneration-related cells is collected. The method of covering is not particularly limited as long as the cell suspension of bone regeneration-related cells does not leak from the container 47. For example, the protrusion 48 of the container 47 can be covered with a rubber stopper.

  Moreover, it is preferable that the container 47 is removable from the bone regeneration composition manufacturing instrument 100, and can collect | recover the cell suspension of the collect | recovered bone regeneration related cell to the projection part 48 by centrifugation. In this case, it is preferable that the lid attached to the protruding portion 48 is such that the cell suspension of the bone regeneration-related cells does not leak out even by the centrifugation operation.

  The material and shape of the container 47 are not particularly limited, but a container having the same material and shape as the container 26 described above can be used.

  As shown in FIG. 3 (b), the support enclosing body 50 has a needle 51 (connecting portion) attached to a container 53 enclosing the support 7. The needle 51 is hollow inside and can pass the cell suspension of bone regeneration-related cells.

  The support enclosure 50 configured as described above allows the protrusion 51 of the container 47 and the support enclosure 50 to communicate with each other by inserting and penetrating the needle 51 into the lid of the projection 48 of the container 47. A cell suspension of bone regeneration-related cells can be transferred from 48 to the support inclusion body 50.

  The bone regeneration-related cells that have flowed from the container 47 through the needle 51 into the support enclosure 50 are mixed with the support 7 in the container 53 of the support enclosure 50 to produce a bone regeneration composition.

  Further, as shown in FIG. 3B, the needle 51 of the support enclosure 50 is covered with rubber 52. When the needle 51 is inserted into the lid of the protrusion 48 of the container 47, the rubber 52 is penetrated by the needle 51. According to such a configuration, the protruding portion 48 of the container 47 and the support enclosure 50 can be communicated with each other while substantially maintaining the closed system.

  In this embodiment, the inside of the container 47 is preferably a negative pressure, and more preferably a vacuum. Since the support 51 is covered with the rubber 52, the support enclosure 50 can keep the inside of the container 47 at a negative pressure, preferably a vacuum.

  Further, if the inside of the container 47 is a negative pressure, preferably a vacuum, when the support enclosure 50 and the protrusion 48 of the container 47 are communicated with each other, an excessive amount of cell recovery solution does not flow in, and sedimentation occurs. Only the bone regeneration-related cells concentrated in this manner can be preferentially flowed into the container 53 of the support inclusion body 50.

  The method for recovering the bone regeneration composition produced from the support inclusion body 50 is not particularly limited. For example, the bone regeneration composition can be recovered from the support inclusion body 50 using a syringe or the like. In addition, after the support enclosing body 50 is separated from the container 47 and the rubber 52 is removed, an external pressure is applied to the container 53, whereby the bone regeneration composition can be pushed out and collected through the needle 51. Thus, when recovering the bone regeneration composition by applying external pressure to the container 53, the bone regeneration composition may be directly transplanted into the diseased part via the needle 51.

<II. Method for producing bone regeneration composition>
The method for producing a bone regeneration composition according to the present invention is a method for producing a bone regeneration composition comprising bone regeneration-related cells involved in bone regeneration and a support that supports the bone regeneration-related cells.

  The method for producing a bone regeneration composition according to the present invention can be preferably carried out using the above-described bone regeneration composition production device according to the present invention.

  Therefore, although the following description demonstrates embodiment which manufactures a bone regeneration composition using the bone regeneration composition manufacturing instrument mentioned above, this invention is not limited to this. That is, the present invention includes a method for producing a bone regeneration composition using the same principle.

  Specifically, the method for producing a bone regeneration composition according to this embodiment includes a step of capturing bone regeneration-related cells with the cell separator 1 (hereinafter also referred to as “cell capture step”), and a cell separator 1. The captured bone regeneration-related cells are caused to flow out of the cell separator 1 by injecting the cell recovery liquid into the cell separator 1, and the cell recovery liquid containing the bone regeneration-related cells that have flowed out of the cell separator 1 is mixed with the mixing container. 6 (hereinafter referred to as “cell recovery step”) and a step of concentrating bone regeneration-related cells in the mixing container 6 and then mixing the bone regeneration-related cells and the support 7 (hereinafter referred to as “mixing step”). Also called).

  Further, a step of injecting a washing solution into the cell separator 1 to wash the cell separator 1 (hereinafter also referred to as “washing step”) is included between the cell capturing step and the cell recovery step. Also good.

  Hereinafter, the cell capture step, the washing step, the cell recovery step, and the mixing step will be described in detail.

[Cell capture step]
In the cell capturing step, bone regeneration-related cells are captured by the cell separator 1. Specifically, the bone regeneration-related cell-containing sample is injected into the cell separator 1 using the sample injection syringe 2 shown in FIG.

  Although the said bone regeneration related cell containing sample is not specifically limited, Specifically, a body fluid, the treatment liquid of a biological tissue, etc. can be mentioned, for example.

  In the present specification, “body fluid” is intended to include blood (including peripheral blood and G-CSF mobilized peripheral blood), bone marrow fluid, umbilical cord blood, and the like, including bone regeneration-related cells. The body fluid includes diluted blood (including peripheral blood and G-CSF mobilized peripheral blood), bone marrow fluid, umbilical cord blood, etc .; blood (including peripheral blood and G-CSF mobilized peripheral blood), bone marrow fluid, umbilical cord Also included are cell suspensions prepared by pretreating blood or the like by specific gravity centrifugation using ficoll, percoll, hydroxyethyl starch (HES), bakatinar tubes, lymphopreps, and the like.

  In addition, the “treatment liquid for biological tissue” means a treatment liquid obtained by decomposing biological tissue with an enzyme (enzymatic degradation treatment liquid), a treatment liquid obtained by decomposing biological tissue by crushing (fracture treatment liquid), and rubbing the biological tissue. Decomposed treatment liquid (rubbing treatment liquid), treatment liquid obtained by decomposing living tissue by osmotic extraction (penetration extraction treatment liquid), treatment liquid obtained by degrading living tissue by a liposuction method performed in cosmetic surgery, etc. Treatment liquid). In addition, about the treatment liquid of these biological tissues, the content of the said patent document 5 is also used as reference in this specification.

  In addition, “biological tissue” intends biological tissue other than body fluid containing bone regeneration-related cells.

  In the cell capture step, the method for injecting (in other words, passing through) the bone regeneration-related cell-containing sample into the cell separator 1 using the sample injection syringe 2 is not particularly limited. Specifically, for example, the plunger of the sample injection syringe 2 containing the bone regeneration-related cell-containing sample can be pushed by hand or machine to inject the bone regeneration-related cell-containing sample into the cell separator 1. .

  When the above-described cell capturing step is carried out without using the bone regeneration composition production tool shown in FIG. 1, the bone separator-related cell is contained in the cell separator 1 by natural fall from a bag or the like in which a bone regeneration-related cell-containing sample is pooled. A sample may be injected.

  Also, the syringe containing the bone regeneration-related cell-containing sample is directly connected to the cell separator 1, and the plunger of the syringe is pushed by hand or machine to inject the bone regeneration-related cell-containing sample into the cell separator 1. You can also.

  Furthermore, a pump or the like may be used for injecting the bone regeneration-related cell-containing sample into the cell separator 1.

  In the cell capturing step, the injection speed (in other words, the liquid passing speed) for injecting the bone regeneration-related cell-containing sample into the cell separator 1 is not particularly limited, and depends on the form of the cell separator 1. A suitable injection rate may be used.

  Specifically, for example, when a cell separator filled with a cell separator having a filter structure is used as the cell separator 1, the passage speed of the bone regeneration-related cell-containing sample with respect to the thickness of the cell separator (linear velocity) ) Is preferably 0.1 mm / min to 1000 mm / min, more preferably 0.5 mm / min to 500 mm / min, and still more preferably 1 mm / min to 250 mm / min.

  If the linear velocity is lower than 0.1 mm / min, the processing time tends to be prolonged. On the other hand, when it is higher than 1000 mm / min, there is a tendency that bone regeneration-related cells capable of colony formation are hardly captured by the cell separator 1 due to the flowing water pressure of the bone regeneration-related cell-containing sample.

  However, within the linear velocity range, the bone regeneration-related cells can be efficiently captured without prolonging the processing time.

[Washing process]
In the washing step, the cell separator 1 washing solution is injected to wash the cell separator 1 after the cell capturing step. Specifically, a washing solution is injected into the cell separator 1 using the washing syringe 4 from the same direction as the direction in which the bone regeneration-related cell-containing sample is flowed in the cell capturing step. Thereby, impurities accumulated in the cell separator 1 can be washed away from the cell separator 1.

  The washing solution is not particularly limited as long as it does not negatively affect the cells. Specifically, for example, a solution generally used as an injectable agent such as physiological saline and Ringer's solution, a buffer solution such as a phosphate buffer, a cell culture medium such as an αMEM medium and a DMEM medium, and the like. it can.

  Among these exemplified solutions, physiological saline can be preferably used because it has a small negative influence on cells and has a large track record of use in medical applications.

  In the above washing step, the injection speed for injecting the cleaning liquid into the cell separator 1 (in other words, passing through the liquid) is not particularly limited, and may be an injection speed suitable for the form of the cell separator 1.

  Specifically, for example, when a cell separator filled with a cell separator having a filter structure is used as the cell separator 1, the washing liquid passage speed (linear velocity) with respect to the thickness of the cell separator is set to 0. It is preferably 1 mm / min to 1000 mm / min, more preferably 0.5 mm / min to 500 mm / min, and even more preferably 1 mm / min to 250 mm / min.

  If the linear velocity is lower than 0.1 mm / min, the processing time tends to be prolonged. On the other hand, if it is higher than 1000 mm / min, bone regeneration-related cells capable of colony formation may flow out of the cell separator 1 due to the flowing water pressure of the washing solution.

  However, within the linear velocity range, the treatment time is not prolonged, and the bone regeneration-related cells capable of forming colonies are accumulated in the cell separator 1 without flowing out of the cell separator 1. Contaminants can flow out of the cell separator 1.

  In the washing step, the amount of the washing liquid injected into the cell separator 1 is not particularly limited, and may be appropriately changed according to the volume of the cell separator 1. Specifically, it is preferable to inject 1 to 100 times the amount of the washing liquid of the volume of the cell separator 1, and it is more preferable to inject 10 to 100 times the amount of the washing liquid.

  By injecting the above-mentioned amount of the washing solution, even when the finally obtained bone regeneration composition is administered to the human body for treatment, impurities can be removed to such an extent that the human body is not adversely affected. it can.

[Cell recovery process]
In the cell recovery step, the cell recovery solution is injected into the cell separator 1 using the recovery syringe 5 from the direction opposite to the direction in which the bone regeneration-related cell-containing sample is injected. As a result, the bone regeneration-related cells captured by the cell separator 1 can be discharged from the cell separator 1. Thus, the cell recovery liquid containing the bone regeneration-related cells flowing out from the cell separator 1 can be recovered in the mixing container 6.

  The cell recovery solution is not particularly limited as long as it does not negatively affect the cells. Specifically, for example, a solution generally used as an injectable agent such as physiological saline and Ringer's solution, a buffer solution such as a phosphate buffer, a cell culture medium such as an αMEM medium and a DMEM medium, and the like. it can.

  Among these exemplified solutions, a medium for cell culture and physiological saline can be preferably used since the negative influence on cells is small.

  Moreover, you may add protein to the said cell collection | recovery liquid from a viewpoint of cell protection. Although the said protein is not specifically limited, Specifically, plasma, serum, albumin etc. can be mentioned, for example.

  Furthermore, a substance for increasing the degree of viscosity may be added to the cell collection solution. Although the said substance is not specifically limited, For example, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethyl cellulose etc. can be mentioned.

  By adding such a substance, the recovery rate of the bone regeneration-related cells captured by the cell separator 1 can be improved.

  In the cell recovery step, the injection speed for injecting (in other words, passing through) the cell recovery liquid into the cell separator 1 is not particularly limited, and may be an injection speed suitable for the form of the cell separator 1. Although it is good, it is preferable to inject as fast as possible.

Specifically, for example, the passage speed (linear velocity) of the cell recovery solution with respect to the thickness of the cell separation material is preferably 50 cm / min to 1000 cm / min, and preferably 100 cm / min to 500 cm / min. More preferred.
According to the above flow rate range, the recovery rate of bone regeneration-related cells can be improved.

  When injecting the cell recovery liquid into the cell separator 1 in such a linear velocity range, for example, by pushing the plunger of the recovery syringe 5 containing the cell recovery liquid vigorously using a hand or a machine. The linear velocity can be realized.

  In the cell recovery step, the amount of the cell recovery solution injected into the cell separator 1 is not particularly limited, and may be appropriately changed according to the volume of the cell separator 1. Specifically, it is preferable to inject a cell recovery solution of 1 to 100 times the volume of the cell separator 1, and more preferable to inject a cell recovery solution of 10 to 100 times the volume.

  By injecting the amount of the cell recovery solution, the bone regeneration-related cells captured by the cell separator 1 can be reliably recovered.

  In the method for producing a bone regeneration composition according to the present invention, it is not necessary to amplify the recovered bone regeneration-related cells in the cell recovery step, but it is preferable not to amplify the recovered bone regeneration-related cells. It does not prevent the regeneration-related cells from being amplified. That is, in the method for producing a bone regeneration composition according to the present invention, the bone regeneration-related cells may be amplified in the cell recovery step.

  Specifically, for example, as the cell recovery solution, Dulbecco MEM medium (Nissui), α-MEM medium (GIBCO BRL), MEM medium (Nissui), IMEM medium (Nissui), RPMI-1640 medium The bone regeneration-related cells can be amplified by collecting the bone regeneration-related cells in the mixing container 6 using a culture medium such as (Nissui), and adhering or growing the bone regeneration-related cells in the mixing container 6. it can.

  Moreover, you may add serum to the said culture medium by the density | concentration of 5%-20% as needed.

When a bone regeneration-related cell is amplified by culture, the culture conditions are not particularly limited, and may be set as appropriate according to the type of the bone regeneration-related cell. For example, it can be cultured in an atmosphere of a temperature of 37 ° C. and CO ₂ gas of 5%.

[Mixing process]
In the mixing step, the bone regeneration-related cells and the support 7 are mixed in the mixing container 6 after the cell recovery step.

  In the present invention, the bone regeneration-related cells are preferably concentrated in the mixing container 6 before or after the bone regeneration-related cells and the support 7 are mixed in the mixing step. According to such a configuration, when the bone regeneration composition finally obtained is used for transplantation, the volume of the bone regeneration composition used for transplantation can be easily adjusted to a volume that matches the size of the diseased part. it can.

  The method for concentrating the bone regeneration-related cells in the mixing container 6 is not particularly limited, but <I. It can concentrate by the method demonstrated by the bone regeneration composition manufacturing instrument>.

  The method of mixing the bone regeneration-related cells and the support 7 in the mixing container 6 is not particularly limited, and the bone regeneration-related cells and the support 7 are substantially different depending on the form of the mixing container 6. A method capable of mixing these may be appropriately selected and used.

  Specifically, for example, (1) a method of mixing the bone regeneration-related cells and the support 7 by applying pressure or attraction, and (2) adding the solution to the bone regeneration-related cells and the support 7. (3) A method of electrically mixing bone regeneration-related cells and the support 7 using the charge of the bone regeneration-related cells and the support 7, (4) By applying vibration, The method etc. which mix a bone regeneration related cell and the support body 7 can be mentioned.

  More specifically, the method of pressurizing in the method (1) is not particularly limited. For example, the method may be such that the syringe is pressed through air or the mixing container 6 itself is pressed by hand. By doing so, it can pressurize. Moreover, the method of drawing pressure is not particularly limited, but for example, drawing can be performed using a syringe or a pump.

  In the method (2), for example, a cell suspension (that is, bone regeneration-related cells are contained in the cell recovery solution) discharged from the cell separator 1 into the mixing container 6 containing the granular support 7. By dropping the suspended cell suspension) from the upper part of the mixing vessel 6, the support 7 and the bone regeneration-related cells can be mixed in the cell suspension solution.

  In the above method (3), for example, since the bone regeneration-related cells are negatively charged by positively charging the support 7, they are attracted to each other electrostatically, and the support 7 and the bone regeneration-related Cells can be mixed.

  In the method (4), the method for applying vibration is not particularly limited. For example, vibration can be applied to the container manually or mechanically.

  Since the method for producing a bone regeneration composition according to the present invention has the above-described configuration, a bone regeneration composition containing at least bone regeneration-related cells and a support can be produced.

  In the present invention, since the bone regeneration-related cells can be concentrated in the mixing container 6 in the mixing step, a bone regeneration composition having a high cell concentration of the bone regeneration-related cells can be produced.

Specifically, it is preferable to contain 1 to 5 × 10 ⁹ bone regeneration-related cells, and more preferably 50 to 5 × 10 ⁸ bone regeneration-related cells to 100 mg of the support. It is more preferable that 1 × 10 ² to 5 × 10 ⁸ bone regeneration-related cells are included.

  When the mixing ratio of the bone regeneration-related cells and the support is within the above range, the bone regeneration-related cells and the support can be mixed uniformly.

The bone regeneration composition preferably contains 1 × 10 ⁵ to 5 × 10 ⁹ nucleated cells with respect to 100 mg of the support, and has 1 × 10 ⁶ to 5 × 10 ⁸ nucleated cells. More preferably, it contains a nuclear cell. The nucleated cell is intended to be a cell having a nucleus, and specific examples include mononuclear cells represented by lymphocytes and monocytes, and multinucleated cells represented by granulocytes. .

  According to such a bone regeneration composition, it can be used as it is for transplantation for bone regeneration treatment without any special operation.

  Moreover, the manufacturing method of the bone regeneration composition concerning this invention can be implemented suitably using the bone regeneration composition manufacturing instrument concerning this invention. That is, according to the bone regeneration composition manufacturing device according to the present invention, the bone containing at least 0.1 part by weight to 10 parts by weight of the bone regeneration-related cell suspension with respect to 1 part by weight of the support. A regenerated composition can be produced.

  Accordingly, the present invention includes a method for producing a bone regenerating composition according to the present invention or a bone regenerating composition manufactured with the bone regenerating composition manufacturing device according to the present invention.

  Furthermore, since the bone regeneration composition according to the present invention can be used for transplantation for bone regeneration treatment, a bone regeneration method for regenerating bone using the bone regeneration composition according to the present invention is also included.

  In addition, the bone regeneration method concerning this invention should just reproduce a bone using the bone regeneration composition concerning this invention. Other specific configurations are not particularly limited, and a conventionally known method may be used.

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example, a comparative example, and FIGS. 4-10, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[Example 1: Removal of red blood cells and unnecessary white blood cells using a bone regenerating composition production apparatus]
A bone regenerating composition manufacturing device shown in FIG. 1 was prepared, and the removal rate of red blood cells and the recovery rate of white blood cells were measured when the bone marrow fluid was treated using the bone regenerating composition manufacturing device.

As the cell separator 1, a column (φ20 mm × length 80 mm, volume 1 cm ³ ) in which a non-woven fabric having a filter structure was filled in a container was used. Moreover, the mixing container 6 used the container in which the support body is not arrange | positioned.

Specifically, first, 10 ml of bone marrow fluid was collected from the iliac bone of a beagle dog. Then, the number of red blood cells and white blood cells contained in 10 ml of this bone marrow fluid was measured. As a result, as shown in FIG. 4, the total number of leukocytes in 10 ml of bone marrow fluid was 429 × 10 ⁶ cells. The total number of red blood cells in 10 ml of bone marrow fluid was 72 × 10 ⁹ cells. In FIG. 4, it is described in the column before processing.

  Next, priming treatment was performed by injecting 30 ml of PBS into the cell separator 1 using the washing syringe 4 of the bone regeneration composition production tool. Subsequently, 10 ml of the above bone marrow fluid was injected into the cell separator 1 using the sample injection syringe 2 at a flow rate of 3 ml / min.

  After injection of the bone marrow fluid, 10 ml of PBS (washing solution) was injected into the cell separator 1 at a flow rate of 3 ml / min using the washing syringe 4. In this way, unnecessary cells were washed.

  After washing, 30 ml of DMEM medium (cell recovery solution) was injected into the cell separator 1 using the recovery syringe 5. In this way, 30 ml of DMEM medium containing mesenchymal stem cells was collected in the mixing container 6.

With respect to 30 ml of the cell collection solution containing the obtained mesenchymal stem cells, the red blood cell count and white blood cell count were measured. As a result, as shown in FIG. 4, the total number of leukocytes was 56 × 10 ⁶ cells. The total number of red blood cells was 1.32 × 10 ⁹ cells. In FIG. 4, it is described in the column after processing.

  Based on the above results, the removal rate of red blood cells and the recovery rate of white blood cells were calculated using the above-mentioned bone regenerating composition production device. As a result, as shown in FIG. 4, the white blood cell recovery rate was 13.1%, and the red blood cell removal rate was 98.2%.

The red blood cell removal rate and the white blood cell recovery rate were calculated using the following calculation formulas as shown in FIG.
Leukocyte recovery rate (%) = total number of leukocytes after treatment / total number of leukocytes before treatment × 100
Erythrocyte removal rate (%) = 100−total number of red blood cells after treatment / total number of red blood cells before treatment × 100.

[Example 2: Performance evaluation of a device for producing a bone regeneration composition]
A device treatment was performed in the same manner as in Example 1 except that 3 ml of commercially available human bone marrow fluid (Veritas Co., Ltd.) was used as the bone marrow fluid. At this time, the number of red blood cells and white blood cells contained in 3 ml of bone marrow before treatment were measured.

As a result, the total number of leukocytes in 3 ml of bone marrow fluid was 45 × 10 ⁶ cells. The total number of red blood cells in 3 ml of bone marrow fluid was 10 × 10 ⁹ cells.

Next, the red blood cell count and white blood cell count were measured for 30 ml of the cell recovery solution containing the mesenchymal stem cells obtained. As a result, the total number of leukocytes was 6 × 10 ⁶ cells. The total number of red blood cells was 0.05 × 10 ⁹ cells. That is, the leukocyte recovery rate was 13.3%, and the red blood cell removal rate was 99.5%.

  As a control, a DMEM medium (hereinafter referred to as “control cell suspension”) containing mesenchymal stem cells prepared by density gradient centrifugation using Ficoll (Ficoll method) was used.

  The control cell suspension was prepared according to the following procedure. First, 1 ml of PBS was added to 3 ml of the above bone marrow fluid. Next, 4 ml of bone marrow diluted with PBS was carefully laminated on 3 ml of Ficoll (Ficoll-paquePLUS) placed in a centrifuge tube so as not to disturb the interface.

  Then, it centrifuged at 1400 rpm for 30 minutes at 18 degreeC-20 degreeC. As a result, the bone marrow fluid has a granulocyte and red blood cell layer, a Ficoll layer, a mononuclear cell and platelet layer (hereinafter also referred to as a “mononuclear cell layer”), and a plasma layer in order from the lower layer. Separated.

  Among the above layers, a mononuclear cell layer was collected. 10 ml of PBS was added to the collected mononuclear cells and resuspended. Then, this suspension was centrifuged at 18 to 20 ° C. and 1500 rpm for 10 minutes.

  After centrifugation, the supernatant was removed, and the operation of resuspending the precipitate in 10 ml of PBS and centrifuging under the above conditions was repeated. The cells thus obtained were suspended in 30 ml of DMEM medium to obtain a control cell suspension.

  In the method using the device, the bone regeneration composition could be prepared in 5 minutes, but the preparation of the control cell suspension took as long as 90 minutes. That is, according to the method of the present invention, mesenchymal stem cells could be separated in a shorter time than the conventional method.

  The number of mesenchymal stem cells in the mesenchymal stem cell suspension and the control cell suspension was determined by the following procedure. Specifically, 15 ml, which is half of the cell recovery solution, was seeded in a T-75 flask, and cultured at 37 ° C. for 8 days. After the culture, the medium was removed, crystal violet staining was performed, and the number of colonies was counted. Here, the number of colonies that appeared was defined as the number of mesenchymal stem cells.

  As a result, as shown in FIG. 5A, 169 mesenchymal stem cells were contained in 15 ml of the mesenchymal stem cell suspension (described as a device in FIG. 5A). . On the other hand, 88 mesenchymal stem cells were contained in 15 ml of the control cell suspension (described as Ficoll in FIG. 5A).

Detection of each cell marker in the mesenchymal stem cell suspension and the control cell suspension was performed according to the following procedure. Specifically, first, cultured cells prepared in the same manner as described above were recovered by enzyme treatment (hereinafter referred to as recovered cultured cells). Next, the collected cultured cells were dispensed into 8 pieces of 1 × 10 ^5, and 10 μL of the following antibody was added thereto. Using this as a sample, the positive rate was measured with a flow cytometer (FACSCalibur, Becton Dickinson).

  As a result, as shown in FIG. 5 (b), the recovered cultured cells (described as the device in FIG. 5 (b)) showed 90% or more of all mesenchymal stem cell markers of CD105, CD73, CD90, and CD166. The positive rate was detected. On the other hand, the positive rate of CD166 was as low as 80% or less in the control-collected cultured cells (described as Ficoll in FIG. 5B).

  The positive rate of CD31, which is a vascular endothelial cell marker, CD133 and CD34, which are hematopoietic stem cell markers, and CD45, which is a leukocyte marker, was as low as 4% or less in both the recovered cultured cells and the control recovered cultured cells.

  As described above, according to the method of the present invention, it was revealed that mesenchymal stem cells can be separated more efficiently than in the conventional method.

  Furthermore, using the collected cultured cells, the ability to form a lumen, the ability to differentiate into bone, and the ability to differentiate into cartilage were evaluated.

  For the lumen forming ability, the recovered cultured cells were suspended in Medium 199 medium, and the following factors were added to the cell suspension to a predetermined concentration (3% FBS, 50 ng / ml VEGF, 10 ng / ml b-FGF). . Evaluation was performed by culturing the cell suspension using Matrigel (BD Bioscience Biotec). The result is shown in FIG.

  For the bone differentiation ability, first, the recovered cultured cells were suspended in αMEM medium, seeded on a 12-well tissue culture plate, and cultured for 24 hours. Next, the following factors were added to the αMEM medium at a predetermined concentration to prepare a bone differentiation induction medium (15% FBS, 10 mM β-GP, 0.1 mM dexamethasone, 50 μg / ml ascorbic acid). After culturing for 24 hours, the medium was replaced with bone differentiation-inducing medium cells, and further culturing was continued, and evaluation was performed by staining with alizarin. The result is shown in FIG.

  Further, for cartilage differentiation ability, first, the recovered cultured cells were suspended in DMEM medium and centrifuged to prepare a pellet. Next, the following factors were added to the DMEM medium to a predetermined concentration to prepare a cartilage differentiation induction medium (0.1 mM dexamethasone, 50 μg / ml ascorbic acid, 6.25 ng / ml insulin, 6.25 ng / ml transferrin). 6.25 ng / ml selenious acid, 1.25 mg / ml bovine serum albumin, 5.35 mg / ml linolenic acid, 10 ng / ml TGF-β3, 40 μg / ml proline). After removing the supernatant of the pellet, the culture was performed by adding a cartilage differentiation-inducing medium, and the cells were evaluated by staining with Alcian blue. The result is shown in FIG.

  As a result, as shown in FIGS. 5C to 5E, the mesenchymal stem cells contained in the mesenchymal stem cell suspension have both lumen forming ability, bone differentiation ability, and cartilage differentiation ability. It was.

  As a result, it has been clarified that the bone regeneration composition producing device according to the present invention can rapidly separate mesenchymal stem cells having both lumen forming ability, bone differentiation ability, and cartilage differentiation ability.

[Example 3: Characterization of mesenchymal stem cells separated by a bone regeneration composition production tool]
Using the same method as in Example 1, mesenchymal stem cells were isolated from two beagle dogs. DMEM medium containing mesenchymal stem cells isolated from each beagle dog was seeded on 3 petri dishes (φ60 mm) so that the number of cells per 2 × 10 ⁴ cells was 37 ° C. For 2 weeks. Thereafter, the number of colonies formed was counted, and the average value of the three petri dishes was calculated.

  In addition, the control cell suspension prepared from the bone marrow fluid each extract | collected from the two beagle dogs using the same method as Example 2 was used for control.

  As a result, as shown in FIGS. 6 (a) and 6 (b), the mesenchymal stem cells (described as “device” in FIG. 6) separated by the bone regenerating composition-producing apparatus of the present invention The colony-forming ability was higher than that of mesenchymal stem cells in suspension (described as “Ficoll” in FIG. 6).

  Next, the morphology of mesenchymal stem cells separated using a bone regenerating composition production device was observed with a microscope. Specifically, bone marrow fluid was collected from one beagle dog, and cultured cells were prepared by the same method as the colony forming ability described above. The cultured cells were observed with a phase-contrast microscope over time (after 8 days, 10 days, and 16 days after seeding) and photographed. The magnification of the microscope at the time of observation was 100 times.

  As a result, as shown in FIG. 7, the mesenchymal stem cells (described as “device” in FIG. 7) separated by the bone regenerating composition production instrument of the present invention were mesenchymal stem cells ( In FIG. 7, it was described as “Ficoll”) and had a normal form as a mesenchymal stem cell.

Furthermore, the proliferative ability of mesenchymal stem cells isolated using a bone regeneration composition production device was examined. Specifically, bone marrow fluid was collected from one beagle dog, and cultured cells were prepared in the same manner as the colony forming ability described above. The cultured cells were once collected by enzyme treatment and seeded on ⁴ petri dishes (φ60 mm) so as to be 3 × 10 ⁴ cells. After seeding, the cells on the petri dish are collected by enzyme treatment over time (24, 72, 120, 168 hours later), and the number of cells is measured with a hemocytometer to observe the change in the number of cells over time. did.

  As a result, the mesenchymal stem cells (described as “device” in FIG. 7) separated by the bone regenerating composition production instrument of the present invention are mesenchymal stem cells in the control cell suspension (“Ficoll” in FIG. 7). The growth curve was almost the same as that described above.

  Specifically, the logarithmic growth phase was entered about 72 hours after the start of the culture, and the stationary phase was reached about 120 hours after the start of the culture.

[Example 4: Bone repair of canine osteonecrosis model]
(1) Preparation of osteonecrosis model Both forelimbs of a beagle dog having a weight of 13 kg and a age of 24 months were developed from the dorsal side, and the lunar scaphoid was identified. The same bone bone cortex was opened, and the cancellous bone inside was removed as much as possible. The prepared defect was filled with liquid nitrogen and then thawed at room temperature for 10 minutes. This procedure was repeated three times to create an osteonecrosis model.

(2) Collection of bone marrow fluid 10 ml of bone marrow fluid was collected from the iliac bones of dogs with osteonecrosis.

(3) Preparation of transplanted cell fraction Twenty-four non-woven fabrics made of rayon and polyolefin (diameter: 18 mm, opening: 5 μm to 48 μm) are laminated in a 20 mm diameter cylindrical polycarbonate, and the laminated non-woven fabric is covered with a polycarbonate stopper. The cell separation material was sandwiched.

  10 ml of bone marrow fluid collected at a linear velocity of 1.6 mm / min was passed through the cell separation material. 10 ml of physiological saline was passed from the same direction at a linear speed of 1.6 mm / min to wash the cell separation material.

  Next, 30 ml of a cell culture solution (DMEM medium) containing 10% fetal bovine serum was vigorously flowed in the direction opposite to the direction in which the bone marrow fluid was flowed to collect the target cell fraction.

  After the collected cell fraction was centrifuged, the supernatant was removed and 100 μL of PBS was added. This cell suspension was poured into an inclusion body containing 0.16 g of β-TCP (β-tricalcium phosphate) and mixed to obtain a transplanted cell fraction (bone regeneration composition).

(4) Transplantation of transplanted cell fraction (bone regeneration composition) The transplanted cell fraction (bone regeneration composition) prepared on one foot side of the osteonecrosis part prepared in (1) above was injected, and this foot was Example 1). Only the inclusion body containing β-TCP (β-tricalcium phosphate) 0.16 g was injected into the osteonecrosis of the opposite foot, and this foot was referred to as (Comparative Example 1). For each foot, the soft tissue and skin were sutured to complete the operation. Then, the amount of bone formation was measured by micro CT (SMX-100CT-SV3type, Shimazu) 28 days after transplantation.

  As a result, as shown in FIG. 9 (in the figure, Comparative Example 1 is described as βTCP only, Example 1 is described as βTCP + MSC), the bone mass-bone volume ratio (the ratio of bone to the total volume) is In Example 1, it was 0.72, whereas in Comparative Example 1, it was 0.63.

  From this, it became clear that according to the transplanted cell fraction (bone regeneration composition), bone formation is promoted.

  Further, as shown in FIG. 9 and FIG. 10B (in the figure, Comparative Example 1 is described as βTCP only, and Example 1 is described as βTCP + MSC), Comparative Example 1 shows clear crush of the lunar scaphoid bone. recognized.

  Furthermore, histopathologically, in Example 1, as compared with Comparative Example 1, there was more infiltration of osteoclasts and osteoblasts in the osteonecrotic part, and good bone tissue regeneration was confirmed.

  As a result, it was revealed that the transplanted cell fraction (bone regeneration composition) is a bone regeneration composition that can be used for bone regeneration treatment.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  As described above, in the present invention, the bone regeneration-related cells are separated from the sample containing the bone regeneration-related cells involved in bone regeneration, and the support is directly used in the closed system without culturing the bone regeneration-related cells. Mix with. Therefore, a bone regeneration composition that can be used for bone regeneration treatment can be easily and rapidly produced. Therefore, the present invention can be widely used for medical use and pharmaceutical use related to bone regeneration.

FIG. 1 is a fragmentary view showing an apparatus for producing a bone regeneration composition according to an embodiment of the present invention. FIG. 2 is a fragmentary view showing a device for producing a bone regeneration composition according to another embodiment of the present invention. FIG. 3 is a fragmentary view showing an apparatus for producing a bone regeneration composition according to still another embodiment of the present invention. FIG. 4 is a diagram showing the removal rate of red blood cells and the recovery rate of white blood cells by the bone regenerating composition production device in the examples of the present invention. FIG. 5 is a diagram showing the performance evaluation results of the bone regenerating composition production device in the examples of the present invention, (a) is a diagram showing the separation performance, and (b) is a mesenchymal stem cell by the surface antigen. (C)-(e) is a figure which shows the differentiation ability of each mesenchymal stem cell isolate | separated, respectively. FIG. 6 is a diagram showing colony-forming ability of mesenchymal stem cells separated using a bone regeneration composition production device in Examples of the present invention, and (a) and (b) are separated from different beagle dogs. It is a figure which shows the colony formation ability of the mesenchymal stem cell which was done. FIG. 7 is a diagram showing the results of observing, with a microscope, the morphology of mesenchymal stem cells isolated using a bone regenerating composition production device in an example of the present invention. FIG. 8 is a diagram showing a proliferation curve of mesenchymal stem cells separated using a bone regeneration composition production device in the examples of the present invention. FIG. 9 is a diagram showing the results of examining the in vivo therapeutic effect of a bone regeneration composition using a canine lunar osteonecrosis disease model in an example of the present invention. FIG. 10 is a diagram showing the results of examining the in vivo therapeutic effect of a bone regeneration composition using a canine lunar osteonecrosis disease model in an example of the present invention, and (a) shows the results of the bone regeneration composition. It is a figure which shows a therapeutic effect, (b) is a figure which shows the result of a control ((beta) -TCP only) experiment.

Explanation of symbols

1 Cell separator (cell capture unit)
2 Syringe for sample injection (sample injection part)
3 Waste liquid bag (Waste liquid recovery part)
4 Cleaning syringe (cleaning liquid injection part)
5 Recovery syringe (cell recovery fluid injection part)
6 Mixing container (cell recovery part)
7 Support 8 Filter (Filtration part)
9a Mixing part (first space)
9b Reservoir (second space)
26 Container (container)
27 Protruding part 28 Tube (connecting part)
29 Support Encapsulation Solution (Support Encapsulation Part)
47 Container (container)
48 Protrusion 50 Support Enclosure (Support Enclosure)
51 needle (connecting part)
100 Bone regenerating composition manufacturing device

Claims (8)

A bone regeneration composition producing device for producing a bone regeneration composition comprising a bone regeneration-related cell involved in bone regeneration and a support that supports the bone regeneration-related cell,
A cell capturing part for capturing the bone regeneration-related cells;
A cell recovery solution injection unit for injecting a cell recovery solution into the cell capture unit, and causing the bone regeneration-related cells captured by the cell capture unit to flow out of the cell capture unit;
A cell recovery unit for recovering a cell recovery solution containing the bone regeneration-related cells that have flowed out of the cell capture unit,
In the cell recovery part, a support that can be mixed with bone regeneration-related cells and used as a bone regeneration composition is disposed,
In the cell recovery part, the bone regeneration-related cells recovered in the cell recovery part and the support are mixed,
The cell recovery unit includes a first space and a second space separated by a filtration unit,
The support is arranged in the first space,
The filtration unit is for filtering a cell recovery solution containing the bone regeneration-related cells recovered in the first space, and allowing components other than the bone regeneration-related cells to flow into the second space. A bone regenerating composition producing device. A sample injection unit for injecting the sample containing the bone regeneration-related cells into the cell capture unit;
The sample injection part is
2. The sample is injected into the cell capture unit in a direction opposite to a direction in which the cell recovery solution injection unit injects the cell recovery solution into the cell capture unit. Bone regeneration composition manufacturing equipment.   The waste liquid collecting unit for collecting a component that has passed through the cell trapping part of the sample when the sample injecting unit injects the sample into the cell trapping part. The bone regeneration composition manufacturing apparatus as described. A washing liquid injection part for injecting a washing liquid for washing the cell capture part into the cell capture part;
The cleaning liquid injection part is
The said cell collection liquid injection | pouring part inject | pours the said washing | cleaning liquid with respect to the said cell capture | acquisition part in the reverse direction to the direction which inject | pours the said cell collection | recovery liquid with respect to the said cell capture | acquisition part. The bone regeneration composition manufacturing instrument according to any one of the above.   The bone regenerative composition production apparatus according to any one of claims 1 to 4, wherein the cell trapping part includes a cell separation material having a filter structure.   6. The cell separation material according to claim 5, wherein when the sample containing leukocytes and erythrocytes is passed through, 30% to 90% of the leukocytes and 90% or more of the erythrocytes are allowed to pass therethrough. Bone regeneration composition manufacturing device.   The bone regeneration composition-producing device according to any one of claims 1 to 6, wherein the bone regeneration-related cell is a mesenchymal stem cell.   The said support body contains calcium phosphate, a natural polymer material, a synthetic polymer material, a metal material, or the composite material which combined these 2 or more types, The any one of Claims 1-7 characterized by the above-mentioned. Bone regeneration composition manufacturing equipment.