JP5155530B2 - Adult stem cell separation and culture system - Google Patents

Description

  In the present invention, adult stem cells are selectively separated from a cell suspension composed of a plurality of cell types, and the separated cells are cultured in the same apparatus, thereby satisfying the quality and quantity necessary for various purposes. The present invention relates to the provision of a cell separation / culture system that makes it possible to obtain adult stem cells.

  As represented by embryonic stem cells, it is well known that fertilized eggs in the early stages of development have abundant stem cells that have the ability to differentiate into various organs and tissues, but even adults that have completed development. There are stem cells. In recent years, it has become a realistic treatment method to treat diseases by compensating or recovering the functions of organs and tissues by transplanting stem cells present in the adult, so-called adult stem cells, into the living body. However, unlike stem cells derived from early developmental tissues such as embryonic stem cells, adult stem cells have a very low presence rate in tissues and include stem cells to prepare the number of stem cells necessary for treatment. It was necessary to secure a large number of organizations.

  For example, for lower limb ischemic diseases such as obstructive arteriosclerosis and Burger's disease, derived from bone marrow, which contains vascular endothelial progenitor cells using a blood component separation device (eg, Baxter CS3000-Plus) from bone marrow Attempts have been made to isolate and transplant the mononuclear cell fraction. In this case, the device only separates the mononuclear cell fraction from the bone marrow fluid, and not only contains many cells other than the target vascular endothelial progenitor cells, but also effective therapeutic vascular endothelial progenitor cells. It does not have the function of amplifying the number. Therefore, usually more than 500 mL of bone marrow fluid is required for treatment, and the patient is forced to undergo general anesthesia for bone marrow fluid collection.

  In addition, an attempt has been made to amplify the number of cells by culturing adult stem cells collected from a patient and apply them to treatment. Specifically, artificial joint replacement has been attempted in which the biocompatibility of the artificial joint is improved by coating the surface of the artificial joint with a mesenchymal stem cell (derived from a patient's bone marrow), which is one of the adult stem cells. However, in this case, the number of cells is amplified by culturing mesenchymal stem cells so that a sufficient number of cells for treatment can be secured even with an amount of bone marrow fluid that can be collected by local anesthesia. Yes. However, in this case, since the bone marrow fluid is seeded in the culture flask as it is and cultured, nucleated cells such as leukocytes and cells other than stem cells such as erythrocytes in the bone marrow fluid are mixed in a large amount in the culture system. Since cells other than adult stem cells deprive the stem cells of the medium components and culture space that the stem cells should originally consume, the amount of media and culture flasks used is significantly increased compared to normal culture. In addition, since adult stem cells are cultured manually in an open environment using normal culture flasks at this time, problems such as contamination with germs and viruses and cross-contamination of other cells during the culture process There is a risk that will occur. For this reason, in order to prepare cells for treatment, it is necessary to strictly manage the culture environment and to ensure that the above problems do not occur.

  As described above, the method and apparatus for preparing adult stem cells that can be applied to treatment have room for further improvement, and it is possible to prepare as many adult stem cells as necessary for treatment even from a small amount of tissue. In addition, an adult stem cell separation / culture apparatus that can be easily prepared is desired.

Patent Documents 1 and 2 disclose a method for separating and concentrating cells for tissue regeneration (that is, adult stem cells referred to in this patent) using a cell separation filter. In this method, since the recovered tissue regenerative cells are cultured in a culture dish or the like, the risk of contamination is unavoidable, and the purity of the recovered adult stem cells is insufficient, so that the isolated tissue When cultivating cells for regeneration, negative effects of contaminating cells (for example, increase in granulocyte elastase concentration due to granulocyte contamination, growth inhibitory effect due to increase in K (potassium) concentration due to hemolysis of erythrocytes, etc.) There is concern.
Further, Patent Document 3 introduces a cell-containing liquid containing culture raw material cells and cells to be removed into a container filled with a cell capturing material made of a porous material, and causes the cell capturing material to capture the culture raw material cells to be removed. We have proposed a cell separation and culture apparatus that is inexpensive, simple and can be operated in a short time by culturing the entire container after the cells are led out of the container. However, since the separation / cultivation method is originally intended to separate monocytes and differentiate them into dendritic cells, the apparatus cultures the whole vessel in a state in which the culture raw material cells are captured by the cell capture material. When this is applied to the isolation and culture of adult stem cells, the number of cells obtained is considered to be limited to some extent due to the space in which the cells can grow.
JP2002-80377 JP 2002-87971 JP 2000-157261 A

  An object of the present invention is to provide an adult stem cell separation / culture system that makes it possible to easily and efficiently prepare the number of adult stem cells necessary for treatment of a disease from a raw material tissue with limited adults. .

The present inventors have intensively studied to solve the above-mentioned problems. As a result, an adult stem cell separation / cultivation system capable of easily and efficiently preparing adult stem cells by selectively separating adult stem cells from a cell suspension and culturing the separated cells in the same apparatus. Was invented. That is, the present invention is as follows.
· at least,
Means 1 for selectively capturing adult stem cells from a solution containing adult stem cells;
A circuit 2 connected to the inlet side of the means 1 and introducing a treatment liquid containing adult stem cells into the means 1;
A circuit 3 connected to the outlet side of the means 1 for draining the liquid derived from the means 1;
Means 4 for introducing the recovered liquid into means 1;
Means 5 for culturing the cells recovered from means 1;
Adult stem cell isolation and culture system composed of
(2) The means 1 contains a cell separation material capable of substantially passing white blood cells and red blood cells in the liquid to be processed in a container having the liquid to be processed introducing section and the liquid extracting section to be processed. The adult stem cell separation / culture system according to (1), which is a cell separator.
(3) The cell separation material is a cell separation material capable of passing 60% or more of white blood cells in the liquid to be treated and allowing 90% or more of red blood cells in the liquid to be treated to pass through. (2) adult stem cell separation and culture system.
(4) The adult stem cell separation / culture system according to (1), wherein the recovery solution used in the means 4 is an adult stem cell culture solution.
(5) The adult stem cell separation / culture system according to (1), wherein a cell culture bag or a cell culture cassette is used as the means 5.
(6) The adult stem cell separation / culture system according to (1), wherein an automatic culture apparatus is used as the means 5.
(7) A method for isolating and amplifying adult stem cells from a liquid to be treated containing adult stem cells, using the adult stem cell separation / culture system according to any one of (1) to (6).
(8) A cell composition prepared using the method of (7).

  According to the present invention, adult stem cells are selectively separated from a cell suspension composed of a plurality of cell types, and the separated cells are cultured to satisfy adult stem cells that can be used for regenerative medicine / cell medicine in both quality and quantity. It was possible to prepare in a fresh state.

  Hereinafter, the present invention will be described in detail.

The adult stem cell separation / culture system of the present invention includes at least:
1) Means 1 for selectively capturing adult stem cells from a liquid to be treated containing adult stem cells;
2) a circuit 2 connected to the inlet side of the means 1 and introducing a treatment liquid containing adult stem cells into the means 1;
3) a circuit 3 connected to the outlet side of the means 1 and draining the liquid derived from the means 1;
4) means 4 for introducing the recovered liquid into means 1;
5) means 5 for culturing the cells recovered from means 1;
An adult stem cell separation and culture system composed of

  An adult stem cell as used in the present invention is an undifferentiated cell existing in the tissue of an adult (including a young individual such as a child or a fetus) that has completed development, and has a specific function of forming a tissue or an organ. It refers to a cell having the ability to differentiate into a cell having it. Specific examples include pluripotency such as mesenchymal stem cells existing in bone marrow fluid, adipose tissue, placenta, amniotic membrane, and adult pluripotent stem cells reported to exist in bone marrow fluid and umbilical cord blood. In addition to stem cells, hematopoietic stem cells present in bone marrow and peripheral blood, vascular endothelial progenitor cells, myoblasts present in muscle tissue, basal cells present in small intestine crypts, liver, kidney, pancreas, Examples include so-called tissue stem cells that exist in organs and tissues such as the retina and skin, but are not particularly limited as long as they retain the ability to differentiate into functional cells. In addition, for example, in the case of human beings, adults that have completed development include not only adult individuals but also infants immediately after birth.

  The treatment liquid containing adult stem cells in the present invention is not limited in terms of physical and chemical properties as long as it is a liquid containing adult stem cells. However, because the adult stem cells are efficiently captured in the means 1 of the present invention. Desirably, adult stem cells are in a cell suspension state dispersed in a liquid. Specifically, body fluids such as bone marrow fluid, peripheral blood, lymph fluid, umbilical cord blood, semen; biochemically by degrading enzyme treatment such as collagenase from organs or tissues, or ultrasonic treatment, homogenizer, sharp blade, etc. However, it is not limited to these. In addition, the above body fluid and treatment solution may be diluted with an appropriate liquid such as physiological saline or phosphate buffer before being treated with the means 1, and concentrated by an appropriate method such as centrifugation. May be. Specific examples of the method for concentration include, but are not limited to, specific gravity density centrifugation using Ficoll, Percoll, Lymphoprep, hydroxyethyl starch (HES), Bactiner tube, and the like. .

  Means 1 in the separation / culture system of the present invention is a circuit that has a function of selectively capturing adult stem cells from a treatment solution containing adult stem cells, and introduces a treatment solution containing adult stem cells to the inlet side thereof. 2 and the circuit 3 for draining the liquid led out to the outlet side can be connected to each other, but any instrument or device can be used. Because it is simple, a cell separation material that can substantially pass white blood cells and red blood cells in the liquid to be treated including adult stem cells is stored in a container having a liquid to be treated inflow part and a liquid outflow part to be treated. It is preferable that it is a cell separator. In that case, the treatment liquid introduction part of the cell separator corresponds to the circuit 2 and the treatment liquid lead-out part corresponds to the circuit 3. The treated liquid introduction part and the treated liquid lead-out part are aseptically connected to the cell separator.

  The cell separation material accommodated in the cell separator is characterized in that it can substantially pass white blood cells and red blood cells in a liquid to be treated containing adult stem cells. As used herein, “substantially passing leukocytes and erythrocytes in the treatment liquid containing adult stem cells” means that when the treatment liquid containing adult stem cells is passed through the cell separation material, leukocytes in the treatment liquid It means that 30% or more and 80% or more of red blood cells pass through without being captured by the cell separation material. From the viewpoint of the ability to separate adult stem cells, a more preferable cell separation material is one that allows passage of 45% or more of leukocytes in the liquid to be treated and 85% or more of red blood cells, and more preferably, in the liquid to be treated. 60% or more of white blood cells and 90% or more of red blood cells are allowed to pass through. Most preferably, 70% or more of white blood cells in the liquid to be treated and 95% or more of red blood cells are allowed to pass through.

  The physical, chemical, and biochemical properties of the cell separation material are not particularly limited as long as the above-described white blood cell and red blood cell passage rates can be achieved and adult stem cells can be selectively captured. Although there are no specific examples of the shape, aperture, density, and material, the following can be cited.

  The form of the cell separation material is not particularly limited, but it is easy to contact the cell suspension and has a large contact area. Preferably there is. More preferably, it is comprised with a fiber, More preferably, it is a nonwoven fabric as a fiber assembly.

  When the cell separation material is composed of a fiber or an aggregate of fibers, the fiber diameter is preferably in the range of 3 μm to 40 μm from the viewpoint of capture / recovery rate of adult stem cells that are target cells. If the fiber diameter of the fibers constituting the cell separation material is smaller than 3 μm, the passing rate of contaminated nucleated cells such as leukocytes, red blood cells, and platelets is lowered, and the removal efficiency thereof is lowered. On the other hand, if the fiber diameter is larger than 40 μm, the effective contact area is likely to be reduced and a short pass is likely to occur, leading to a decrease in the capture / recovery rate of target cells. In order to increase the interaction between the target cell and the cell separation material and increase the yield, the fiber diameter is more preferably in the range of 5 μm to 35 μm. More preferably, it is the range of 5 micrometers-30 micrometers.

  The mesh size of the cell separation material is preferably 3 μm or more in the minor axis and 120 μm or less in the major axis from the viewpoint of capturing / recovering target cells. If the minor axis of the cell separation material is smaller than 3 μm, the removal efficiency of contaminating nucleated cells such as leukocytes, erythrocytes, and platelets is significantly reduced. If the major axis of the mesh is larger than 120 μm, it becomes difficult to capture adult stem cells as target cells. From the viewpoint of removal efficiency of contaminated nucleated cells such as leukocytes, erythrocytes and platelets, preferably, the minor axis of the aperture is 5 μm or more and the major axis is 80 μm or less. From the viewpoints of removal efficiency, adult stem cell capture / recovery rate, etc., more preferably, the minor axis of the mesh is 5 μm or more and the major axis is 70 μm or less. The term “aperture” as used herein refers to an image analysis device in which a cell separation material is photographed with a scanning electron microscope, and the major and minor diameters of a substantial hole formed by the intersection of two or more different fibers. It is the average value of the values measured at 50 points or more.

The density of the cell separation material is 1.0 × 10 ⁴ g / m ³ to 1.0 × 10 from the viewpoint of removal efficiency of contaminated nucleated cells such as leukocytes, red blood cells and platelets, and capture / recovery rate of target cells. A range of ⁶ g / m ³ is preferred. Further, from the viewpoint of removal efficiency of contaminated nucleated cells such as leukocytes, red blood cells, and platelets, more preferably 2.5 × 10 ⁴ g / m ^{3 to} 7.5 × 10 ⁵ g / m ³ , still more preferably 5.0. in the range of ^{× 10 4 g / m 3 ~5.0} × 10 5 g / m 3. The density referred to here is a value obtained by dividing the weight (g) of the cell separation material by its volume (m ³ ).

  Cell separation material is made of polyolefin such as polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyester, polyvinyl chloride, polyvinyl alcohol, polyvinylidene chloride, rayon, vinylon, polystyrene, acrylic polymer (polymethyl methacrylate, poly Hydroxyethyl methacrylate, polyacrylonitrile, polyacrylic acid, polyacrylic acid ester, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, polyacrylate, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin A material selected from at least one of chitosan, glass, cotton and the like is preferable. More preferably, it is a synthetic or semi-synthetic polymer selected from at least one of polyester, polypropylene, polystyrene, acrylic polymer, rayon, polyolefin, vinylon, polyethylene, nylon, polyurethane and the like.

  When two or more kinds of polymers are combined as the cell separation material, the combination is not particularly limited, and examples thereof include polyester and polyolefin, or rayon and polyolefin, or a combination of polyester, rayon, and vinylon. When the two or more kinds of polymers are combined, the form of the fiber may be a fiber in which one fiber is composed of polymers of different components, or a split fiber in which different components are separated from each other. Moreover, the form which each compounded the polymer which consists of polymer | macromolecule independent from which a component differs 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 made of a polymer alone is bonded to each other. It is not limited.

  In order to further improve the performance of the cell separation material, the material may be hydrophilized. By imparting hydrophilic treatment, non-specific capture of cells other than necessary cells can be suppressed, body fluid can be passed through the cell separation material without unevenness, and recovery efficiency of necessary cells can be improved. Can do. Hydrophilic treatment methods include water-soluble polyhydric alcohols, polymers having hydroxyl groups, cationic groups, and anionic groups, or copolymers thereof (eg, hydroxyethyl methacrylate, dimethylaminoethyl methacrylate, or copolymers thereof). A method of adsorbing a water-soluble polymer such as polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, a method of immobilizing a hydrophilic polymer on a hydrophobic film (for example, chemically immobilizing a hydrophilic monomer on the surface) A method of binding, etc.), a method of irradiating the cell separation material with an electron beam, a method of crosslinking and insolubilizing the hydrophilic polymer by irradiating the cell separation material with water, and a heat treatment of the cell separation material in a dry state. , Method of insolubilizing and immobilizing hydrophilic polymer, Forming hydrophilic polymer and water-insoluble complex A method of treating a cell separation material with a component to be treated, a method of sulfonating the surface of a hydrophobic membrane, a method of forming a membrane from a mixture of a hydrophilic polymer substance such as polyethylene glycol or polyvinylpyrrolidone and a hydrophobic polymer dope, an alkali A method of imparting hydrophilic groups to the membrane surface by treatment with aqueous solution (NaOH, KOH, etc.), A method of immersing a hydrophobic porous membrane in alcohol, treating with a water-soluble polymer aqueous solution, drying, and insolubilizing treatment with heat treatment or radiation And a method of adsorbing a substance having a surface active action.

  Nonionic surfactant, lecithin, polyoxyethylene hydrogenated castor oil, sodium edetate, sorbitan sesquioleate, D-sorbitol, dehydrocholic acid, glycerin, D-mannitol, tartaric acid, propylene Examples include 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. Polyoxyethylene surfactants include polyoxyethylene alcohol ether, polyoxyethylene acyl ester, polyoxyethylene sorbitan acyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene Examples include sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan monooleate and the like. These can be used alone or in combination. In addition, in order to improve the adherence of adult stem cells to a cell separation material, a cell adhesion protein or an antibody against an antigen expressed on a target cell may be immobilized on the cell separation material.

  Examples of cell adhesion proteins include fibronectin, laminin, vitronectin, collagen and the like. As an antibody, when the target cell is a mesenchymal stem cell, an antibody against CD73, CD90, CD105, etc., an hematopoietic stem cell, an antibody against CD34, c-Kit, Sca-1, etc., and a myoblast, Examples include, but are not limited to, antibodies to Myf5 and the like.

  In the present invention, the cell separator when used as the means 1 contains the cell separating material described above, but there is no particular limitation on the form, size, structural material, and the like.

The cell separator may take any form such as a sphere, a container, a cassette, a bag, a tube, a column, and the like. Preferred specific examples include, for example, a capacity of about 0.5 to 1000 ml, and a diameter of about 0. Examples thereof include a transparent or translucent cylindrical container of about 1 to 10 cm, or a square or rectangular shape having a length of about 1 cm to 20 cm and a thickness of about 0.1 cm to 5 cm. Is not limited to this. The cell separator can be made using any structural material. Specific examples include non-reactive polymers, biocompatible metals, alloys, and glass. Non-reactive polymers include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymers; halogenated polymers such as polytetrafluoroethylene, polychlorotrifluoroethylene, copolymers of tetrafluoroethylene and hexafluoropropylene, and polyvinyl chloride; polyamides, polysulfones Polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, and the like. Examples of the metal material useful as the container material include stainless steel, titanium, platinum, tantalum, gold, and alloys thereof, as well as gold-plated alloy iron, platinum-plated alloy iron, cobalt chromium alloy, titanium nitride-coated stainless steel, and the like.
Particularly preferred are materials having sterilization resistance, and specific examples include polypropylene, polyvinyl chloride, polyethylene, polyimide, polycarbonate, polysulfone, and polymethylpentene.

  In the adult stem cell separation / culture system of the present invention, as the means 4 for introducing the recovered solution into the means 1, any instrument or device can be used as long as the object can be achieved. In the case of the cell separator containing the cell, it is preferable that the cell trapped by the cell separation material can be efficiently recovered by applying a high flowing water pressure. Specific examples thereof include pumps that can push out liquids such as plunger pumps, piston pumps, and gear pumps, and devices that can push out liquids contained in cylinders, such as syringes, by pushing the pistons. However, it is not limited to these.

  As the recovery liquid introduced into the means 1 using the means 4, any liquid may be used as long as it does not negatively affect the captured cells. Specifically, those that have been used for medical purposes such as physiological saline and Ringer's solution, media used for cell culture, buffer solutions such as phosphate buffer solution and Hank's salt solution, or serum, plasma Etc. Among them, since the adult stem cells recovered from the means 1 by the means 4 are subsequently cultured in the means 5, a liquid having a function of proliferating adult stem cells, that is, a culture solution of adult stem cells is used as the recovered liquid. Preferably there is. Specific examples of the culture solution of adult stem cells in this case include cell culture media such as D-MEM (Dulbecco modified Eagle medium), IMDM (Iskov modified Eagle medium), RPMI1640 medium, MCDB133 medium, ASF medium, and the like. It is not limited to these. In addition, in order to enhance the proliferation ability and differentiation ability of the recovered stem cells, the above culture medium is added to biological components such as serum and plasma, bFGF (basic fibroblast growth factor), TGF-β (transforming growth factor − β), protein factors such as insulin, transferrin, laminin, fibronectin, retinoic acid, ascorbic acid, various amino acids, various sugars, 5-azacytidine, 2-mercaptoethanol, sodium selenite, hydrocortisone, dexamethasone Molecular substances may be added. Furthermore, in order to avoid contamination of germs in the stem cell culture process, antibiotics and antifungal agents such as kanamycin, gentamicin, penicillin, streptomycin, polymyxin B, vancomycin, amphotericin B may be added to the above liquid.

  In the means 4, in order to improve the recovery rate of the adult stem cells trapped by the cell separation material, the consistency of the recovery solution used may be increased. The substance added for that purpose is not particularly limited, and examples thereof include albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, and hydroxyethylcellulose.

  In the adult stem cell separation / culture system of the present invention, as means 5 for culturing the recovered cells, as long as adult stem cells contained in the recovered cells can be cultured and the number of the cells can be amplified, Any instrument or device can be used, but since the operation is simple and the risk of contamination and cross-contamination is low, a cell culture bag, a cell culture cassette, or an automatic cell culture device can be used. preferable.

  The cell culture bag refers to a bag-like container composed of a flexible (flexible) structural material in which cells can be stored and cultured. The cell culture bag can be used on a flat table such as a tray installed in the incubator, hung on a suitable frame, or shaken using a shaker. There is no. Cell culture bag structural materials include polyolefin resins such as polypropylene, polyethylene, and ethylene propylene copolymers, elastomers such as styrene butadiene thermoplastic resins, and Teflon (registered) in terms of gas permeability and biocompatibility. Trademark), silicone rubber and the like are preferable, but are not particularly limited. Preferred specific examples include Nipro culture bags (made by Nipro), Optitice, X-FOLD, LifeCell (made by Baxter) and the like, but are not limited thereto.

  The cell culture cassette refers to a container made of an inflexible structural material in which cells can be stored and cultured. The shape of the cell culture cassette is not particularly limited, such as circular, rectangular, cylindrical, prismatic, spherical, etc., but is preferably a flat rectangle because many cell culture cassettes can be installed in a limited space. The structural material of the cell culture cassette is not particularly limited, but gas permeable polystyrene, polycarbonate, and the like are preferable because inflexibility can be ensured and the cell and medium in culture are highly visible because it is transparent. . The cell culture cassette can be used on a flat table such as a tray installed in the incubator, stacked in an appropriate rack, or shaken using a shaker. There is no limit. Preferable specific examples include OptiCell (manufactured by BioCrystal) and CLINIcell (manufactured by MABIO), but are not limited thereto.

  The automatic culture apparatus refers to an apparatus that can automatically or semi-automatically replace all or part of the operation required for cell culture usually performed by a method with a machine or an instrument. Preferable specific examples include Aastrom Replicacell System (manufactured by Aastrom Bioscience), and culture apparatuses disclosed in JP-A-2004-344128, JP-A-2004-89095, JP-A-2001-275659, and the like. Is not to be done.

  In the adult stem cell separation / culture system of the present invention, the means 5 is aseptically connected to the means 1 until the adult stem cell separation / recovery operation. In the subsequent culturing step, the means 5 is separated from the means 1. It is also possible to carry out the cultivation.

Next, a preferred method for separating and culturing adult stem cells from a treatment solution containing adult stem cells, which is performed using the adult stem cell separation / culture system of the present invention, will be described with reference to FIG.
FIG. 1 shows an example of an embodiment of the present invention, but the present invention is not limited to this.

  The liquid to be treated containing adult stem cells prepared by any method is temporarily stored in the liquid tank 2-1. In this case, since the prepared liquid to be processed is transferred in a closed system environment, the liquid tank 2-1 is provided with an inlet capable of aseptically connecting the means for preparing the liquid to be processed and the liquid tank to be processed. It is preferable that A liquid to be treated containing adult stem cells stored in the liquid tank to be treated is passed through a line 2-2 corresponding to the circuit 2 and a cell separator 1-1 corresponding to the means 1. The means for passing the liquid to be processed to the cell separator 1-1 is not particularly limited, and a device such as a pump may be used, or the liquid may be passed by natural fall due to gravity. The flow rate of the liquid to be processed to the cell separator 1-1 is not particularly limited, but when expressed by the passing speed (linear speed) of the liquid to be processed with respect to the thickness of the cell separating material, 0.1 mm / min. It is preferably in the range of 1,000 mm / min. When the linear velocity is lower than 0.1 mm / min, the treatment time is prolonged, and when it is higher than 1,000 mm / min, adult stem cells are hardly captured by the separation material due to the flowing water pressure of the liquid to be treated. A more preferable linear velocity is 0.5 mm / min to 500 mm / min, and even more preferably 1 mm / min to 250 mm / min.

When the treatment liquid containing the adult stem cells is passed through the cell separator 1-1, the adult stem cells in the treatment liquid are captured by the cell separation material, and the erythrocytes other than the captured erythrocytes and adult stem cells are nucleated. Unnecessary components such as cells, plasma components, enzymes, and buffers used for dilution are transferred to the liquid tank 3-1 through the line 3-2 corresponding to the circuit 3.
By passing the liquid to be treated containing adult stem cells through the cell separator 1-1, the adult stem cells are captured by the cell separator, but at the same time, some red blood cells and nucleated cells remain in the cell separator. There is a possibility. In order to remove these unnecessary cells, the washing solution may be passed through the cell separator 1-1. In this case, it is preferable that the cleaning liquid inlet is upstream of the liquid inlet to be processed which has passed through the cell separator 1-1. The washing solution used here is not particularly limited as long as it does not negatively affect adult stem cells. Specific examples thereof include physiological saline, Ringer's solution, serum, plasma, medium used for cell culture, phosphorus Examples include general buffer solutions such as acid buffers. It is preferable to use physiological saline because it has a small negative effect on adult stem cells and has a track record of being used for medical purposes. The washing liquid may be passed through the cell separator 1-1 using the circuit 2 in the same manner as the liquid to be treated containing adult stem cells, or a circuit different from the circuit 2 may be provided to the cell separator 1-1. It may be passed through. In any case, it is preferable to use conditions for passing the cleaning liquid in accordance with the case where the liquid to be treated is passed through the cell separator 1-1.

When the washing solution is passed through the cell separator, unnecessary cells other than the adult stem cells remaining in the cell separation material leave the cell separation material and pass through the line 3-2 to the liquid tank 3-1 together with the washing solution. Be transported.
The adult stem cells captured by the cell separation material are passed through the cell separator 1-1 through the line 4-2 using the cell recovery liquid injector 4-1 corresponding to the means 4. Is transferred to the cell culture device 5-1 through the line 5-2 and collected. The line 4-2 may be connected to a line 2-2 located on the upstream side of the cell separator 1-1 via a three-way cock, or the line 3-2 located on the downstream side may be connected to the three-way cock 4-3. However, since it is highly possible that adult stem cells are unevenly distributed upstream in the cell separator 1-1, it is preferably connected to the line 3-2 located on the downstream side.

  By passing the cell recovery solution through the cell separator 1-1, the adult stem cells recovered in the cell culture device 5-1 are directly cultured in the cell separator 5-1. At this time, it is preferable to use a liquid having a function of proliferating stem cells, such as a medium for stem cell culture, as the cell recovery solution, since it can be cultured as it is after transfer. When the cell recovery solution is passed through the cell separator 1-1 via 3-2, the line 5-2 is connected to the line 2-2 via the three-way stopcock 5-3. When the recovered solution is passed through the cell separator 1-1 via the line 2-2, the cell recovered solution is connected to the line 2-2, the cell culture device is connected to the line 3-2, and the cell culture device And then cultured. In this case, the former is preferable from the viewpoint of the recovery rate of adult stem cells.

The recovered adult stem cells are cultured using the cell incubator 5-1, and are amplified to the required number of cells. At this time, they may be amplified while maintaining the properties as adult stem cells, or appropriate. Differentiation may be induced in specific cells or tissues. The cell composition amplified while maintaining the properties as an adult stem cell and the cell composition induced to differentiate into a specific cell or tissue are also included in the scope of the present invention. The adult stem cells amplified to the required number of cells in the cell culture device 5-1 may be further cultured outside the cell culture device 5-1, or outside the cell culture device 5-1. Differentiation induction can also be performed with.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

Example 1
An example of producing an adult stem cell separation / culture apparatus corresponding to the adult stem cell separation / culture system of the present invention, separating mesenchymal stem cells, which are a kind of adult stem cells, from bone marrow fluid, and amplifying them is shown below. The apparatus used in the present example is the one shown in FIG. 1, and a liquid to be treated containing adult stem cells is prepared in a liquid tank 2-1, and cell culture is performed between the cell separator 1-1 and the line 2-1. And the unnecessary cell collection container 3-1 downstream of the cell separator 1-1, and the cell collection liquid injection device 4-1 between the cell separator 1-1 and the unnecessary cell collection container 3-1. There is a system. As the cell separator 1-1, a split fiber nonwoven fabric (density = 1.3 × 10 ⁵ g / m ³ , fiber diameter = ³ to 12 μm) formed in a cylindrical tube having an inner diameter of 1 cm provided with an entrance and exit. By laminating 12 sheets of openings = 5 to 50 μm (minor axis to major axis) and thickness 5.5 × 10 ⁻⁵ m), and sandwiching the upper and lower sides of the nonwoven fabric with a stopper having an outer diameter of 1 cm, an inner diameter of 7 mm and a height of 5 mm A cell separator with a non-woven fabric immobilized thereon was used.

  Bone marrow used as a treatment solution containing adult stem cells was prepared by the method described below. Anesthesia was performed by injecting ketalal and seractal by intramuscular injection into domestic pigs weighing approximately 30 kg, and then adding nembutal by intravenous injection. Heparin was put in advance in a 10 ml syringe so as to be about 20 IU / ml, and bone marrow fluid was collected from the iliac using a 15 G puncture needle. Next, heparin was added to the collected bone marrow pool to a final concentration of 50 IU / ml, and the mixture was thoroughly mixed by inversion to obtain bone marrow fluid.

First, the cell separation material was washed with a physiological saline solution of about 6 times its volume. Place 5 ml of the prepared bone marrow fluid in the liquid tank 2-1, set the bone marrow injection speed to 0.5 ml / min (linear speed 13 mm / min) with the roller clamp attached to the circuit, and then connect the cells through the line 2-2. The liquid to be treated introduced into the separator 1-1 and derived from the cell separator was sampled from the line 3-2, and the blood cell count was measured with an automatic blood cell counter (Sysmex K-4500). Next, 10 ml of physiological saline was flowed at the same flow rate through the line 2-2 from the same direction, thereby washing and removing red blood cells, white blood cells, and platelets. Next, 5 ml of a cell culture solution (α-MEM medium) containing 15% of fetal bovine serum is placed in the cell recovery solution injection device 4-1, and the line 4-2 and the line 3 from the direction opposite to the direction in which the bone marrow fluid has flowed. -2 is used to collect the target cell fraction in a cell culture device 5-2 (using CLINIcell (manufactured by MABIO)), and the number of blood cells in the collected solution is determined. It was determined with an automatic blood cell counter. The passing rate of blood cells was determined by dividing the number of various cells after passing through the cell separator 1-1 by the number of blood cells before passing through the cell separator 1-1. The cell recovery rate was determined by dividing the number of cells in the recovery solution by the number of blood cells before passing through the cell separator 1-1. As a result, the passing rate of red blood cells and platelets was 95% or more, and the passing rate of white blood cells was about 75%. The recovery rates were 0.8% for red blood cells, 4% for platelets, and about 18% for white blood cells. From this, it was shown that erythrocytes and platelets were almost removed by this cell separation filter. Next, the cell culture device 5-2 was aseptically disconnected and cultured in a CO ₂ incubator at 37 ° C. The medium was changed every 2-3 days, and the number of colonies that appeared by staining the colonies with crystal violet 10 days after the start of the culture was measured. As a result, the number of appearing colonies was 85 / cassette.

Next, cartilage formation evaluation was performed using the cells on the 10th day cultured in the cell culture device 5-2. Cells cultured and amplified by the above method were washed once with 20 ml of DMEM-high glucose medium manufactured by GIBCO BRL, and collected by centrifugation (1000 rpm, 10 min, 4 ° C.). GIBCO BRL manufactured DMEM-high glucose medium to promote the induction of cartilage differentiation (TGFβ3 human recombinant final concentration 10 ng / ml: Funakoshi, dexamethasone final concentration 100 nM: Sigma, ascorbic acid phosphate final concentration 50 μg / ml: WAKO, Sodium pyruvate final concentration of 100 μg / ml, L-proline final concentration of 40 μg / ml: Cosmo Bio) plus ITS (insulin, transferrin, selenium, bovine serum albumin) 1/100 volume of commercial stock solution The added medium was suspended in a mesenchymal cell concentration of 4 × 10 ⁵ cells / ml. 0.5 ml of this cell suspension was taken and placed in a 15 ml falcon tube. Thereafter, centrifugation (1000 rpm, 10 min, 4 ° C.) resulted in pelleting of the cells, but the lid of the tube was loosened and cultured in a 37 ° C., 5% CO ₂ incubator for 3 weeks. During this period, the medium was changed twice a week. After completion of the culture, the spherical cell mass was collected, fixed with formalin for tissue fixation, and stained with toluidine blue, which is a cartilage matrix stain. As a result of observing the tissue section under a microscope, a metachromatism in which the cartilage matrix was stained purple was observed (FIG. 2).

(Reference Example 1)
The cartilage matrix forming ability was evaluated in the same manner as in Example 1 except that the cartilage differentiation inducing factor (above) was not added to the medium. As a result, no metachromatism (metachromosis) in which the cartilage matrix was stained purple was observed (FIG. 2).

(Comparative Example 1)
2 ml of bone marrow fluid collected in the same manner as in Example 1 was mixed (2 times diluted) with 2 ml of phosphate buffer PBS (−). Next, 3 ml of Ficoll paque plus (Amersham) was added to a 15 ml centrifuge tube, and 4 ml of the diluted bone marrow fluid prepared previously was layered on the upper layer of the solution. The mononuclear cell fraction containing adult stem cells was recovered by centrifugation at 1400 rpm for 30 min at room temperature. About 10 ml of PBS (-) was added and the cells were washed at 1300 rpm for 5 minutes. Next, about 10 ml of PBS (−) was similarly added, and the cells were washed again at 1200 rpm for 5 minutes. The rewash solution was suspended in 2 ml of PBS (−), the blood cell count was measured, and the cell recovery rate was determined. In addition, colony appearance rate and heterochromatability were observed in the same manner as in Example 1. As a result, the recovery rate of red blood cells was 1%, the recovery rate of platelets was 8%, and the recovery rate of white blood cells was 83%. The number of colonies that appeared was 79 / cassette.

The results relating to the adult stem cell recovery performance are shown in Table 1, and the observation results of metachromaticity are shown in FIG. From the above results, by using the adult stem cell separation / culture system of the present invention, the present culture system has mesenchymal stem cell separation performance superior to the Ficoll separation method currently used as a method for separating adult stem cells. It was found that the cells proliferated in the above have the ability to differentiate into cartilage.

An example of the adult stem cell isolation culture system of this invention. Results of chondrogenesis evaluation of recovered cells performed in Example 1 and Reference Example 1

Explanation of symbols

1 Portion corresponding to means 1 1-1 Cell separator (device main body)
2 Device inlet side unit 2-1 Liquid tank 2-2 Line 3 Device outlet side unit 3-1 Liquid tank 3-2 Line 4-1 Cell recovery liquid injection device 4-2 Line 4-3 Three-way stopcock 5-1 Cell culture 5-2 Line 5-3 Three-way stopcock

Claims (7)

at least,
Means 1 for selectively capturing adult stem cells from a solution containing adult stem cells;
A circuit 2 connected to the inlet side of the means 1 and introducing a treatment liquid containing adult stem cells into the means 1;
A circuit 3 connected to the outlet side of the means 1 for draining the liquid derived from the means 1;
Means 4 arranged on the outlet side of means 1 for introducing the recovered liquid into means 1;
Means 5 for culturing cells recovered from means 1 disposed on the inlet side of means 1;
Adult stem cell isolation and culture system composed of A cell separator in which means 1 contains a cell separation material capable of substantially passing white blood cells and red blood cells in a liquid to be processed in a container having a liquid to be processed introducing section and a liquid extracting section to be processed. The adult stem cell separation / culture system according to claim 1, wherein: The cell separation material is a cell separation material capable of passing 60% or more of white blood cells in the liquid to be treated and allowing 90% or more of red blood cells in the liquid to be treated to pass through. Item 3. The adult stem cell separation / culture system according to Item 2. The adult stem cell separation / culture system according to claim 1, wherein the recovery solution used in the means 4 is an adult stem cell culture solution. The adult stem cell separation / culture system according to claim 1, wherein the means 5 is a cell culture bag or a cell culture cassette. The adult stem cell separation / culture system according to claim 1, wherein an automatic culture apparatus is used as the means 5. A method for separating and amplifying an adult stem cell from a treatment liquid containing the adult stem cell using the adult stem cell separation / culture system according to any one of claims 1 to 6.