JPWO2013069503A1 - Stem cell isolation method - Google Patents

Abstract

Provided are a method for efficiently separating stem cells from a body fluid such as bone marrow fluid while suppressing contamination, and a stem cell obtained by a general method. The following steps (1) to (2): (1) a step of adding a divalent cation chelating agent to a body fluid containing heparin, and (2) separation of adherent stem cells from the body fluid using a cell separation filter A method for separating stem cells, characterized in that the steps of:

Description

The present invention relates to a method for separating stem cells from a body fluid such as bone marrow fluid or umbilical cord blood using a cell separation filter, and a stem cell-containing fraction obtained by the treatment method.

It is known that body cells such as bone marrow fluid and umbilical cord blood have stem cells having properties that can be differentiated into various cells such as bone, cartilage, muscle, and fat (Patent Document 1, Non-Patent Document 1). Non-patent document 2, Non-patent document 3). Stem cells have the ability to differentiate into a wide variety of cells and organs, and a method for efficiently separating and amplifying stem cells from body fluids is extremely important from the viewpoint of the development of regenerative medicine. It has been reported that only about 1 in 10,000 to 1,000,000 stem cells are present in bone marrow fluid in adults (Non-patent Document 4), and various methods for recovering the stem cell fraction after separation and concentration have been studied. .

For example, Pittenger et al. Have found that differentiation precursor cells into fat, cartilage, and bone cells are present in a fraction having a specific gravity of 1.073 using the Ficoll-pack fractionation method, which is a density gradient centrifugation method (non-native). Patent Document 4). Sekiya et al. Have also attempted to differentiate into cartilage using cells separated by specific gravity by Ficoll pack fractionation (Non-patent Document 5). Wakiya et al. Have tried to obtain cells of fractions other than erythrocytes using dextran and to differentiate into cartilage using these cells (Non-patent Document 6). However, the separation method using Ficoll or dextran requires an operation of washing the cells several times using a centrifuge to separate the separation liquid from the cells, and the operability is complicated. There is a problem that cells are damaged and there is a risk of contamination due to operation in an open system.

Thus, as a closed separation method, a stem cell separation method using a device using a nonwoven fabric has been reported (Patent Document 2). This method is a method of separating stem cells by capturing cells with non-woven fibers, and also captures some white blood cells and platelets at the same time in addition to the stem cells, so that unnecessary cells other than the stem cells are mixed at the time of recovery. . When a large amount of bone marrow fluid is processed, clogging of the nonwoven fabric occurs due to the capture of leukocytes and platelets, and the fluid permeability is impaired.

As a drug to be added to the cell fraction in addition to Ficoll and the like, it has been reported that a divalent cation chelating agent is used as a cell protecting / stabilizing agent (Patent Document 3). However, there is no description or suggestion about suppression of clogging of the nonwoven fabric.

As described above, in view of the complexity of operation and the risk of contamination due to an open system, a method of separating stem cells from body fluids using a cell separation filter is preferable, but unnecessary leukocytes and platelets in the stem cell-containing fraction are preferred. Problems such as contamination and clogging of the nonwoven fabric when a large amount of bodily fluids are treated are listed, and no solution has yet been found and practically used.

International Publication No. 01/083709 Pamphlet International Publication No. 07/046501 Pamphlet JP 2007-89532 A

Pliard A. et al .: J. Cell Biol. 130 (6): 1461-72 (1995) Mackay A. M. et al .: Tissue Engineering 4 (4): 415-428 (1998) Angele P. et al .: Tissue Engineering 5 (6): 545-553 (1999) Pittenger. Et al .: Science 284: 143-147 (1999) Sekiya. Et al .: Developmental Biology 7 (99): 4397-4402 (2002) Wakitani. Et al.: Osteoarthritis Research Society International (2002) 10,199-206

An object of the present invention is to solve the problems in the method of separating stem cells from body fluid using a cell separation filter. Specifically, the present invention provides a treatment method for suppressing clogging of a cell separation filter when treating a large amount of body fluid. Furthermore, the processing method which reduces the ratio of the unnecessary white blood cell and platelet in a stem cell containing fraction is provided.

The present inventors have conducted intensive research on a method for efficiently separating stem cells from body fluids using a cell separation filter from the viewpoint of safety and operability. As a result, surprisingly, by adding a divalent cation chelating agent at a specific concentration to the body fluid that has been anticoagulated by adding heparin, adhesion of leukocytes and platelets, which are contaminated cells, is suppressed. The present inventors have found a method capable of efficiently collecting sex stem cells and completed the present invention.

That is, the present invention includes the following steps (1) to (2): (1) a step of adding a divalent cation chelating agent to a body fluid containing heparin, and (2) a cell separation filter from the body fluid. The present invention relates to a method for separating stem cells, wherein the step of separating adherent stem cells is performed in this order.

It is preferred that the stem cell is a mesenchymal stem cell.

The body fluid is preferably bone marrow fluid.

The divalent cation chelating agent is preferably citric acid.

It is preferable that the addition concentration of citric acid is 0.028 to 3.69% (w / v).

It is preferable that the contamination rate of platelets in the cell suspension containing the separated stem cells is 20% or less.

The cell separation filter is preferably made of polyolefin or / and rayon.

The cell separation filter is preferably made of a nonwoven fabric.

It is preferable that the density of the nonwoven fabric is 2.1 × 10 ^{5 to} 4.9 × 10 ⁵ g / m ³ and the fiber diameter is 3 to 40 μm.

The cell separation filter is preferably filled in a container having a body fluid inlet and outlet.

The present invention also relates to stem cells isolated by the above method.

By using the stem cell separation method of the present invention, stem cells can be efficiently separated from body fluids while suppressing the contamination of contaminating cells, so that stem cells can be collected with high purity. Stem cells obtained by this method can be used for regenerative medicine and cell medicine, and can be used as they are without being proliferated, or can be proliferated and used. When the cell is used as it is without being proliferated, the volume of the cells can be reduced, which is useful when transplanting to a site having a limited volume. When proliferating, it is possible to proliferate the cells in a concentrated state, which leads to cost reduction and simplification of the procedure through a scale-down of the culture container and the like during the proliferation.

Hereinafter, the present invention will be described in detail. The stem cell separation method of the present invention includes the following steps (1) to (2): (1) a step of adding a divalent cation chelating agent to a body fluid containing heparin, and (2) a cell separation filter. The step of separating adherent stem cells from the body fluid is performed in this order.

The body fluid in the present invention refers to bone marrow fluid, umbilical cord blood, blood (including peripheral blood, G-CSF mobilized peripheral blood), menstrual blood, and the like, including stem cells. Also included are those obtained by liquefying biological tissue such as adipose tissue by enzyme treatment, and those obtained by resuspending cells obtained from the liquefied product in a buffer such as physiological saline or a cell culture medium.

In addition, as the body fluid in the present invention, a diluted solution of the above-mentioned body fluid, Ficoll, Percoll, hydroxyethyl starch (HES), bacterin tube, lymphoprep, or the like is used. Also included are cell suspensions prepared by treatment.

The stem cell in the present invention refers to a cell characterized in that it grows by attaching to a culture dish and has differentiation ability. Specifically, it refers to pluripotent cells such as mesenchymal stem cells, bone marrow stromal cells, and pluripotent stem cells. A mesenchymal stem cell refers to a cell that is isolated from a body fluid, has the ability to repeat self-proliferation, and can differentiate into a downstream cell lineage. This mesenchymal stem cell is differentiated into osteoblasts, chondrocytes, vascular endothelial cells, cardiomyocytes, etc., or fat, periodontal tissue constituent cells such as cement blasts, periodontal ligament fibroblasts, etc. by differentiation inducing factors. It is a cell. Bone marrow stromal cells refer to all adherent cell components in bone marrow cells except immature and mature blood cells. A pluripotent stem cell refers to a cell that can be differentiated into a nerve cell or a hepatocyte by a differentiation-inducing factor, but is not limited thereto.

The contaminating cells in the present invention include cells other than stem cells in body fluids, that is, leukocytes, platelets and the like.

The heparin in the present invention refers to heparins such as heparin and low molecular weight heparin. If the concentration of heparin in the body fluid is too low, coagulation may occur in the body fluid. If the concentration in the body fluid is too high, the body fluid is diluted and a long time is required for cell separation. From these viewpoints, the concentration of heparin in the body fluid is preferably 0.1 to 500 IU / mL, more preferably 1 to 200 IU / mL, further preferably 5 to 100 IU / mL, and most preferably 10 to 100 IU / mL. Stem cells can be separated by adding heparin to the collected body fluid, and when heparin is contained in commercially available body fluids, it is also possible to separate the stem cells without adding heparin separately.

The divalent cation chelating agent in the present invention is EDTA-2 sodium, EDTA-2 potassium, EDTA-3 potassium, ammonium oxalate, potassium oxalate, citric acid, monosodium citrate, disodium citrate, tricitrate citrate Although sodium etc. are mention | raise | lifted, the solution containing the citric acid and its salt marketed as a blood preservation solution from a viewpoint of availability is preferable.

Examples of the solution containing citric acid, which is a divalent cation chelating agent, and salts thereof include ACD (acid-citrate-dextrose) -A solution, ACD-B solution, CPD (Citrate-Phosphate-Dextrose) solution, and the like. If the concentration of citric acid added to the body fluid is too low, the effect of suppressing contamination of contaminated cells may be diminished, and if the concentration added to the body fluid is too high, the pH of the body fluid may become acidic or stem cells Capture may be reduced. From these viewpoints, the concentration of citric acid added to the body fluid is preferably 0.01 to 10% (w / v), more preferably 0.02 to 5% (w / v), and 0.028 to 3.69. % (W / v) is more preferable, and 0.028 to 2.51% (w / v) is most preferable.

The material of the cell separation filter in the present invention is polyolefin such as polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, polyhydroxy Ethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton What consists of at least 1 sort (s) chosen from etc. is preferable. More preferably, a synthetic polymer composed of at least one selected from polyester, polystyrene, acrylic, rayon, polyolefin, vinylon, nylon, polyurethane and the like can be mentioned.

When two or more synthetic polymers are combined, the combination is not particularly limited, and preferred examples include polyester and polypropylene; rayon and polyolefin; or a combination of polyester, rayon and vinylon.

As a form of the fiber when combining two or more kinds of synthetic polymers to form fibers, a fiber in which one fiber is made of a synthetic polymer of different components, or a divided fiber in which different components are separated from each other may be used. Moreover, the form which each compounded the fiber which consists of synthetic polymers with different components individually may be sufficient. The term “composite” as used herein is not particularly limited, and examples thereof include a form constituted by a state in which two or more kinds of fibers are mixed, or a form in which a form composed of a single synthetic polymer is bonded together. It is not limited to these.

The form of the cell separation filter in the present invention is not particularly limited, and examples thereof include a porous body having a communicating pore structure, a fiber assembly, and a woven fabric. From the viewpoint of achievement as a cell separation filter (for example, leukocyte removal filter), it is preferably composed of fibers, and more preferably non-woven fabric.

In the present invention, the clogging of the cell separation filter means that the pressure on the inlet side of the cell separation column packed with the cell separation filter is 70 mmHg or more. If the pressure on the inlet side is 70 mmHg or more, problems such as a decrease in the recovery rate of the target stem cells occur, which is not preferable. The pressure on the inlet side where clogging of the cell separation filter does not become a problem is preferably 60 mmHg or less, more preferably 50 mmHg or less, and further preferably 40 mmHg or less.

The density of the cell separation filter is desirably 2.1 × 10 ^{5 to} 4.9 × 10 ⁵ g / m ³ from the effect of removing red blood cells and the like and the recovery rate of the target stem cells. When the density of the cell separation filter is less than 2.1 × 10 ⁵ g / m ³ , the rate at which the target cells pass through the cell separation filter is doubled or more, and the recovery rate of the target cells is reduced. When exceeding 4.9 * 10 < ⁵ > g / m < ³ >, it will become easy to produce clogging with a foreign material, and there exists a possibility that a collection rate may fall. More preferably recovery of objective cell ^{2.6 × 10 5 ~4.9 × 10 5} g / m 3, more preferably from ^{3.2 × 10 5 ~4.8 × 10 5} g / m 3. The density can be adjusted by the material of the separation filter, fiber diameter, porosity, etc., but can also be adjusted by compressing the same separation filter.

The fiber diameter of the cell separation filter is preferably 3 to 40 μm from the viewpoint of improving the recovery rate of target cells. If it is thinner than 3 μm, the interaction with leukocytes increases and the removal efficiency of red blood cells, leukocytes, and platelets decreases. When the thickness is larger than 40 μm, the effective contact area is likely to be reduced and a short pass is likely to occur, and the recovery rate of target cells is likely to be reduced. In order to increase the interaction between the target cell and the cell separation filter and the recovery rate, it is more preferably 3 to 35 μm, still more preferably 5 to 35 μm, and still more preferably 5 to 30 μm. When the cell separation filter is composed of fibers, the fiber diameter can be obtained, for example, by taking a photograph of the cell separation filter with a scanning electron microscope, measuring the length of any 30 points, and averaging. . When the cell separation filter is a porous body or the like, the fiber diameter means the average width of the resin portion (portion that is not a hole) in the cross-sectional portion of the porous body, and is measured in the same manner as described above.

In order to further improve the performance of the cell separation filter, the cell separation filter may be hydrophilized. By carrying out the hydrophilization treatment, non-specific capture of cells other than the target stem cells can be suppressed, and body fluid can be passed through the cell separation filter without bias, thereby improving the recovery efficiency of necessary cells. Hydrophilic treatment methods include water-soluble polyhydric alcohols, polymers having hydroxyl groups, cationic groups, anionic groups, or copolymers thereof (for example, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, or copolymers thereof). Method of adsorbing; Method of adsorbing water-soluble polymer (polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, etc.); Method of immobilizing hydrophilic polymer on hydrophobic membrane (eg, chemically bonding hydrophilic monomer to the surface) Method); electron beam irradiation method; hydrophilic polymer is crosslinked and insolubilized by irradiating the cell separation filter with water in a water-containing state; hydrophilic polymer is insolubilized and fixed by heat treatment in a dry state. A method; a method of sulfonating the surface of a hydrophobic membrane; a hydrophilic polymer; A method of forming a film from a mixture with an aqueous polymer dope; A method of imparting a hydrophilic group to the film surface by treatment with an alkaline aqueous solution (NaOH, KOH, etc.); A hydrophobic porous membrane is immersed in alcohol and then treated with a water-soluble polymer aqueous solution And a method of insolubilizing with heat treatment or radiation after drying; a method of adsorbing a substance having a surface active action, and the like.

Examples of the hydrophilic polymer include polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, ethylene-vinyl alcohol copolymer, polyhydroxyethyl methacrylate, polysaccharide (cellulose, chitin, chitosan, etc.), water-soluble polyhydric alcohol, and the like.

Examples of the hydrophobic polymer include polystyrene, polyvinyl chloride, polyolefin (polyethylene, polypropylene, etc.), acrylic, urethane, vinylon, nylon, polyester, and the like.

Nonionic surfactants, lecithin, polyoxyethylene hydrogenated castor oil, sodium edetate, sorbitan sesquioleate, D-sorbitol, dehydrocholic acid, glycerin, D-mannitol, tartaric acid, Examples include propylene glycol, macrogol, lanolin alcohol, and methylcellulose. Nonionic surfactants are broadly classified into polyhydric alcohol fatty acid esters and polyoxyethylenes. Examples of the polyhydric alcohol fatty acid ester surfactant include glyceryl stearate ester, sorbitan fatty acid ester, sorbitan acyl ester and the like. Examples of the polyoxyethylene surfactant include polyoxyethylene alcohol ether, polyoxyethylene acyl ester, polyoxyethylene sorbitan acyl ester, polyoxyethylene sorbitan monolaurate and the like. These can be used alone or in combination.

Furthermore, in order to improve the adherence of the target adherent stem cells to the cell separation filter, cell adherent proteins and antibodies against specific antigens expressed on the target stem cells are added to the cell separation filter. It may be fixed to. Examples of cell adhesion proteins include fibronectin, laminin, vitronectin, collagen and the like. Examples of antibodies include, but are not limited to, antibodies against CD73, CD90, CD105, CD166, CD140a, CD271, and the like. Examples of immobilization methods include general protein immobilization methods such as cyanogen bromide activation method, acid azide derivative method, condensation reagent method, diazo method, alkylation method, and crosslinking method. It can be used arbitrarily.

The cell separation filter may be filled into a container having a body fluid inlet and outlet and used as a cell separation column. At this time, the cell separation filter may be filled into a container without being compressed, You may compress and fill a container. The shape of the cell separation filter is not limited as long as the above conditions are satisfied. Preferable specific examples include those obtained by filling a cell separation column container shown below with a non-woven cell separation filter having a thickness of about 0.1 to 5 cm in a packed state. In this case, the thickness of the cell separation filter in a packed state is preferably 0.1 cm to 5 cm, but more preferably 0.15 to 4 cm from the viewpoint of the target stem cell recovery rate and the removal efficiency of red blood cells and the like. 0.2 to 3 cm is more preferable. When the thickness of the cell separation filter is less than the above thickness, the cell separation filter may be laminated to satisfy the conditions.

Alternatively, the cell separation filter may be wound into a roll and filled in the cell separation column container. When used in a roll form, the body fluid may be treated from the inside to the outside of the roll to capture the target stem cells, or conversely, the body fluid flows from the outside to the inside of the roll. The target stem cells may be captured.

There are no particular limitations on the form, size, and material of the container used for the cell separation column. The form of the container may be any form such as a sphere, a container, a cassette, a bag, and a tube. Preferable specific examples include, for example, a cylindrical container having a capacity of about 0.1 to 400 mL and a diameter of about 0.1 to 15 cm, a square or a rectangle having a length of about 0.1 to 20 cm, and a thickness of 0.1. Examples include a rectangular columnar container of about 1 to 5 cm, but the present invention is not limited to these.

The container can be made using any structural material. Specific examples of the structural material include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymers; polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, halogenated polymers such as polyvinyl chloride; polyamides, Examples thereof include polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, and polymethylpentene. Metal materials (biocompatible metals, alloys) useful as container materials include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt-chromium alloy, and nitride. Examples thereof include titanium-coated stainless steel. Particularly preferred is a material having sterilization resistance, and specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycardinate, polysulfone, polymethylpentene and the like.

The cell separation column is formed by filling a cell separation filter in a container having an inlet and an outlet for body fluid, and further, a cell separation filter is filled in a container having an inlet and an outlet for cell washing solution and cell recovery solution. In addition, a cell separation filter may be filled in a container provided with a culture bag for culturing the collected cells as they are.

Specifically, the cell separation column has a body fluid inlet and an outlet for discharging the body fluid that has passed through the cell separation filter. Further, the cell separation column is independent of the inlet or the inlet side other than the inlet. And a washing liquid inlet for washing non-adherent cells remaining in the cell separation filter, and independently captured on the outlet or the outlet side other than the outlet. It is preferable that a cell recovery liquid inlet for recovering cells is provided for flowing the cell recovery liquid from the direction opposite to the flow of body fluid and washing liquid.

Stem cells, which are useful cells, can be captured by passing body fluid through the cell separation filter. After passing the body fluid, the washing solution is passed in the same direction as the passage of the body fluid, so that the erythrocytes remaining on the cell separation filter can be washed away. Then, it is possible to separate the stem cells by flowing the cell recovery solution, and the recovery rate is improved by flowing the recovery solution in the direction opposite to the direction in which the body fluid and the washing solution are flowed.

In the present invention, it is preferable to recover 50% or more of stem cells in body fluid, more preferably 60% or more, and even more preferably 70% or more.

In the present invention, the platelet contamination rate in the cell suspension containing the separated stem cells is preferably 20% or less, and more preferably 14% or less. If the platelet contamination rate in the collected cell suspension is too high, the purity of the target stem cells tends to decrease, and the culture efficiency after separation tends to decrease.

Examples of the method for separating stem cells of the present invention will be described below, but the present invention is not limited thereto.

1) preparation of body fluids;
Body fluid is collected from a human or other animal, and heparin is added in an amount of about 0.1 to 500 IU / mL and mixed. Next, about 0.01 to 10% (w / v) of citric acid is added and mixed.

2) Delivery of body fluids;
The body fluid prepared in 1) is passed through the cell separation column composed of the cell separation filter, and the cell fraction containing the target stem cells is captured by the cell separation filter. When the liquid is passed, the liquid may be naturally dropped from the container containing the body fluid through the liquid feeding circuit or may be passed by a pump. Alternatively, a syringe containing body fluid may be directly connected to the column and injected by manually pushing the syringe. When the liquid is passed by a pump, a flow rate of about 0.1 to 100 mL / min is mentioned.

3) Washing;
A washing solution is passed through the cell separation column from the inlet side to wash away cells such as red blood cells. The cleaning liquid may be sent by natural fall through a circuit or may be passed through a pump. About 0.1-100 mL / min is mentioned as a flow rate in the case of letting liquid flow with a pump. The washing amount varies depending on the capacity of the cell separation column, but it is preferable to wash with about 1 to 100 times the column volume. Examples of the cell washing solution include physiological saline, Ringer's solution, medium used for cell culture, and general buffers such as phosphate buffer, and physiological saline is preferable from the viewpoint of safety.

4) Recovery;
A cell collection solution is put into the cell separation column from the direction opposite to the direction in which the body fluid and the washing solution are flowed (outlet side), and the fraction containing the target stem cells is collected. When the cell recovery liquid is injected into the cell separation column, it can be realized by putting the cell recovery liquid in a syringe or the like in advance and pushing the plunger of the syringe with a hand or the like. The amount of collected liquid and the flow rate vary depending on the column volume, but it is preferable to inject a cell collected solution of about 1 to 100 times the column volume at a flow rate of about 0.5 to 20 mL / sec.

The cell recovery solution is not particularly limited as long as it is an isotonic solution, and examples thereof include those that have been used as injections such as physiological saline and Ringer's solution, buffer solutions, and cell culture media.

Further, in order to increase the recovery rate of the stem cells captured by the cell separation filter, the viscosity of the cell recovery solution may be increased. For this purpose, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethyl cellulose, collagen, hyaluronic acid, gelatin and the like can be added to the cell recovery solution, but the present invention is not limited thereto.

The stem cell-containing fraction washed and recovered according to the present invention may be further cryopreserved. From the point that damage to cells can be reduced, it is better to cryopreserve using liquid nitrogen. In addition, cryopreserved cells can be thawed and transplanted into humans or animals, used for research, or cultured again.

A pharmaceutical composition can be produced according to the present invention. By the stem cell recovery method described above, a pharmaceutical composition can be produced by concentrating the stem cells and mixing the concentrated cells with a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include coagulants, nutrient sources such as vitamins, antibiotics, and the like.

Stem cells obtained using the cell separation filter can be proliferated and provided in an undifferentiated state, or can be used without being proliferated. In addition, cells that are transplanted into cartilage injury patients, cells that are transplanted into bone disease patients, cells that are transplanted into myocardial disease patients or vascular disease patients, and patients who have damaged nerve tissue are induced by differentiation induction with a differentiation inducer or the like. Although it can be used as a cell, it is not limited to these. The differentiation inducer in this case is not particularly limited, but examples of the differentiation inducer for cartilage include dexamethasone, TGFβ, insulin, transferrin, ethanolamine, proline, ascorbic acid, pyruvate, selenium, and the like. Examples of the differentiation inducer include dexamethasone, β-glycerophosphate, vitamin C, ascorbate and the like, and examples of the differentiation inducer to the myocardium include EGF, PDGF, 5-azacytidine, and the like. Includes EGF, bFGF, bHLH, and the like, and examples of the blood vessel differentiation inducer include bFGF, VEGF, and the like.

In addition, stem cells obtained using the cell separation filter can be used as therapeutic cells. Furthermore, the therapeutic cells can be applied to various diseases to treat them. Specific treatment targets include stem cell fatigue disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation, heart disease, immunodeficiency, Crohn's disease, etc. It is not limited to these.

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

(Example 1)
(1) Preparation of bodily fluid ACD-A solution was added to human bone marrow fluid (Fresh Whole Bone Marlow manufactured by AllCells, containing 50 IU / mL of heparin) at a ratio of bone marrow fluid: ACD-A solution = 72: 1 (citric acid (Final concentration: 0.028%), and the mixture was thoroughly mixed by inversion.

(2) Production of Cell Separation Column A cylindrical polycarbonate tube having an inner diameter of 2.2 cm and a length of 0.9 cm provided with an inlet / outlet, and a nonwoven fabric composed of rayon and polyolefin as a cell separation filter (mesh = 95 (g / m ² ), fiber diameter = 15 ± 9 μm, density 3.8 × 10 ⁵ (g / m ³ )) is punched out into a circle with a diameter of 2.2 cm, 24 sheets are compressed and laminated, and the upper and lower sides are sandwiched between stoppers to give cells. A cell separation column with a separation filter fixed thereto was prepared.

(3) Cell separation performance evaluation The nonwoven fabric was washed with 50 mL of physiological saline. Next, 3 mL of bone marrow fluid was passed through the syringe pump at a flow rate of 6 mL / min. Next, 30 mL of physiological saline was flowed from the same direction at the same flow rate to wash and remove red blood cells and the like. Next, 50 mL of a cell culture solution (GIBCO α-MEM 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.

<Measurement of blood cell contamination>
The number of white blood cells, red blood cells, and platelets contained in the collected cell suspension was measured with an automatic blood cell counter (Sysmex K-4500). The cell contamination rate was determined by dividing this result by the number of various cells before passing through the column. As a result, the leukocyte contamination rate = 62%, the red blood cell contamination rate = 3%, and the platelet contamination rate = 14%.

<Column clogging evaluation>
The pressure on the column inlet side was measured with a pressure gauge when the bone marrow fluid was passed. This result was used as an index for clogging (the clogging of the column impairs the permeability of the bone marrow fluid and increases the pressure on the column inlet side). This evaluation was performed at the time of 30 mL bone marrow fluid treatment (Example 4 and Comparative Example 2).

<CFU-F assay>
The number of mesenchymal stem cells isolated was analyzed by CFU-F assay. First, a cell culture solution (GIBCO α-MEM medium) containing fetal bovine serum 10% was added to 1/3 amount of the collected cell suspension (equivalent to 1 mL of bone marrow fluid treatment) and transferred to a polystyrene petri dish (diameter 10 cm). Culturing was performed at 5 ° C. in a 5% CO ₂ environment. Next, after 9 days of culture, colonies were stained with crystal violet-methanol solution, and the number of colonies that appeared was measured. The number of recovered colonies was 361.

(Example 2)
The cell contamination rate and the number of colonies were the same as in Example 1, except that the amount of ACD-A solution added was bone marrow fluid: ACD-A solution = 100: 15 (final citrate concentration: 0.28%). Asked. As a result, leukocyte contamination rate = 44%, erythrocyte contamination rate = 3%, platelet contamination rate = 0%, and the number of recovered colonies = 391.

(Example 3)
The cell contamination rate and the number of colonies were the same as in Example 1 except that the amount of ACD-A solution added was bone marrow fluid: ACD-A solution = 2: 1 (final citrate concentration: 0.72%). Asked. As a result, leukocyte contamination rate = 44%, erythrocyte contamination rate = 3%, platelet contamination rate = 0%, and the number of recovered colonies = 401.

Example 4
The same method as in Example 1 except that the amount of bone marrow fluid treated was 30 mL and the amount of cell suspension used in <CFU-F assay> was 1/30 amount (corresponding to 1 mL of bone marrow fluid treated). The cell contamination rate and the number of colonies were determined. As a result, leukocyte contamination rate = 16%, erythrocyte contamination rate = 1%, platelet contamination rate = 5%, and the number of recovered colonies = 342. Also, when the pressure at the column inlet side was measured with a pressure gauge as an indicator of clogging during passage of bone marrow fluid (the more the column was clogged, the permeability of the bone marrow fluid was impaired and the pressure at the column inlet side increased). The pressure was 31 mmHg.

(Example 5)
Pharmacopoeia sodium citrate was dissolved in physiological saline and added to porcine bone marrow fluid (containing 50 IU / mL of heparin) so that the final concentration of citric acid was 0.05%, followed by thorough inversion. For the preparation and evaluation of the cell separation column, the cell contamination rate and the number of colonies were determined in the same manner as in Example 1. As a result, the white blood cell contamination rate = 64.4%, the red blood cell contamination rate = 3%, the platelet contamination rate = 0%, and the number of recovered colonies = 301.

(Example 6)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 5 except that the final citric acid concentration was 0.28%. As a result, leukocyte contamination rate = 57.2%, erythrocyte contamination rate = 3%, platelet contamination rate = 0%, and the number of recovered colonies = 309.

(Example 7)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 5 except that the final citric acid concentration was 0.50%. As a result, leukocyte contamination rate = 57.2%, erythrocyte contamination rate = 3%, platelet contamination rate = 0%, and the number of recovered colonies = 283.

(Example 8)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 5 except that the final citric acid concentration was 1.00%. As a result, the leukocyte contamination rate = 57.2%, the red blood cell contamination rate = 3%, the platelet contamination rate = 0%, and the number of recovered colonies = 251.

Example 9
The cell contamination rate and the number of colonies were determined in the same manner as in Example 5 except that the final citric acid concentration was 2.51%. As a result, the leukocyte contamination rate = 64.4%, the erythrocyte contamination rate = 6%, the platelet contamination rate = 0%, and the number of recovered colonies = 262.

(Example 10)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 5 except that the final citric acid concentration was 3.69%. As a result, leukocyte contamination rate = 50%, erythrocyte contamination rate = 3%, platelet contamination rate = 0%, and the number of recovered colonies = 175.

(Comparative Example 1)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 1 except that the column treatment was performed without adding the ACD-A solution to the body fluid. As a result, the leukocyte contamination rate = 62%, the red blood cell contamination rate = 3%, the platelet contamination rate = 21%, and the number of recovered colonies = 363.

(Comparative Example 2)
The cell contamination rate and the number of colonies were determined in the same manner as in Example 4 except that the column treatment was performed without adding the ACD-A solution to the body fluid. As a result, the leukocyte contamination rate = 35%, the red blood cell contamination rate = 1%, the platelet contamination rate = 29%, and the number of recovered colonies = 265. When the pressure at the column inlet side was measured with a pressure gauge as an indicator of clogging during passage of bone marrow fluid, the maximum pressure was 92 mmHg.

The above results are summarized for each bone marrow fluid treatment amount and are shown in Table 1.

In Examples 1 to 3 and 5 to 10, since a decrease in the white blood cell and / or platelet contamination rate can be confirmed as compared with Comparative Example 1, the concentration is 0.028 to 3.69% (w / v). It can be seen that by adding an acid to the bone marrow fluid, contamination of leukocytes and / or platelets can be suppressed. In addition, it was shown that contamination of leukocytes and / or platelets can be suppressed without suppressing the recovery of mesenchymal stem cells by setting citric acid to 2.51% (w / v) or less.

In Example 4 where the amount of bone marrow fluid treatment is large, a decrease in the mixing rate of leukocytes and platelets was remarkably confirmed as compared with Comparative Example 2, and the number of recovered colonies was higher in Example 4 than in Comparative Example 2. The effect of adding citric acid was remarkably shown. Furthermore, the maximum pressure on the column inlet side was significantly lower in Example 4 than in Comparative Example 2, and it was confirmed that clogging of the filter was suppressed.

Claims (11)

The following steps (1) to (2):
(1) a step of adding a divalent cation chelating agent to a body fluid containing heparin, and (2) a step of separating adherent stem cells from the body fluid using a cell separation filter,
Are performed in this order. A method for separating stem cells. The method according to claim 1, wherein the stem cells are mesenchymal stem cells. The method according to claim 1, wherein the body fluid is bone marrow fluid. The method according to claim 1, wherein the divalent cation chelating agent is citric acid. The method according to claim 4, wherein the addition concentration of citric acid is 0.028 to 3.69% (w / v). The method according to any one of claims 1 to 5, wherein the contamination rate of platelets in the cell suspension containing the separated stem cells is 20% or less. The method according to any one of claims 1 to 6, wherein the cell separation filter comprises polyolefin or / and rayon. The method according to any one of claims 1 to 7, wherein the cell separation filter comprises a nonwoven fabric. The method according to claim 8, wherein the density of the nonwoven fabric is 2.1 × 10 ^{5 to} 4.9 × 10 ⁵ g / m ³ and the fiber diameter is 3 to 40 μm. The method according to any one of claims 7 to 9, wherein the cell separation filter is filled in a container having a body fluid inlet and an outlet. The stem cell isolate | separated by the method in any one of Claims 1-10.