JP2004018504A - Cell preservation container - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medical utensil for performing a cell transplantation treatment simply and easily, i.e., enabling anybody to perform the preservation of the cells and injection of them into a living body easily. <P>SOLUTION: This cell preservation container is provided by forming its inside surface with a material to which the cells hardly adhere, e.g. a material bonded or coated with a hydrophilic material or hydrophobic material, and therefore the conventional peeling off operation of the cells or cleaning operation becomes unnecessary. Also, at least one part of the container is formed by a gas permeable material for enabling the gas exchange required for the survival of the cells and the long period preservation of the cell, and further, enabling the injection operation by taking a syringe like form. With such the constitution, it becomes possible to provide the cell preservation container capable of performing the culturing/preservation /injection into the living body by simple operations. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cell storage container that can store cells therein and, when the cells are needed, can immediately remove the cells from the container and use them without performing a troublesome operation. More specifically, the present invention relates to a cell storage container devised so that when cells are used for treatment in regenerative medicine or the like, the cells can be immediately injected into a living body.
[0002]
[Prior art]
In recent years, due to advances in cell biology and cell engineering, regenerative medicine research in which cells (stem cells) having pluripotency and self-renewal ability extracted from a living body are transplanted to a patient and treated is rapidly progressing. This is a treatment method for repairing and recovering a target tissue (organ) by transplanting a stem cell obtained by inducing differentiation into a target cell into a deficient part of a living tissue or a site responsible for a disease. At present, it is still in the stage of basic research, but some clinical trials have begun. In such a case, generally, undifferentiated cells or differentiated cells obtained by culturing cells alone or in a dedicated container in the presence of a scaffold are transplanted to a target site by surgery.
[0003]
[Problems to be solved by the invention]
The above-described cell transplantation method requires a surgical operation, and therefore places a heavy burden on patients. Therefore, a method of directly injecting cells into a living body has been studied. In particular, injection of chondrocytes or their precursor cells into joints, injection of nerve cells or their precursor cells into the brain, injection of cardiomyocytes or their precursor cells into the heart, etc. can be effective treatments. In the case of performing such treatment, in the conventional method, cells that have proliferated and adhered to the container wall are treated with trypsin, EDTA, or the like to be detached, and after a step such as washing, a predetermined amount is collected in a syringe. It will be injected into the living body.
[0004]
However, in such a method, not only the operation steps are many and troublesome, but also a series of operations must be performed by a skilled person in a clean environment so as to prevent contamination from the surroundings. For this reason, it is difficult to carry out cell transplantation treatment in facilities other than well-equipped facilities.
[0005]
An object of the present invention is to provide a medical device that can easily carry out cell transplantation treatment. That is, the present invention provides a medical device that allows anyone to easily store and inject cells into a living body.
[0006]
[Means for Solving the Problems]
In the present invention, the above-mentioned problem has been solved by storing the grown cells in a container whose inner surface is formed of a material to which the cells are unlikely to adhere. That is, the present invention is a cell storage container in which the inner surface of the container is formed of a material to which cells are unlikely to adhere. By forming the inner surface of the container with a material to which the cells do not easily adhere, the cells do not adhere to the container wall surface during storage, and the operation of peeling the cells from the container wall surface becomes unnecessary. The expression "the cells are hard to adhere" means that the cells do not adhere at all during the storage of the cells, or that they adhere only to such an extent that they are easily peeled off even if they adhere.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Although the inner surface of the cell storage container of the present invention is formed of a material to which cells are unlikely to adhere, it is convenient if the container has a function of a syringe because it can be directly injected into a living body. In addition, it is preferable to form a part or the whole of the container with a gas-permeable material, since oxygen and carbon dioxide necessary for survival of cells can be permeated even in a sealed state, and the cells can be stored for a long period of time.
[0008]
【Example】
Examples of the material that is difficult to adhere to cells used in the present invention include those whose surface is formed of a hydrophilic material or a hydrophobic material and those whose surface has a negative charge. The hydrophilic material preferably has a contact angle with water of 50 degrees or less, and the hydrophobic material preferably has a contact angle with water of 100 degrees or more. Examples of preferred hydrophilic materials include acrylamide polymers, methacrylamide polymers, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, cellulose, dextran, hyaluronic acid, glycosumming glycans, proteoglycans, Materials in which carrageenan, protein, and the like are bonded to the surface of the base material by a method such as graft copolymerization or chemical reaction, or the surface is coated. Examples of the hydrophobic material include fluororesins such as polytetrafluoroethylene and tetrafluoroethylene-hexafluoropropylene copolymer and silicone resins. Examples of the material having a negative charge on the surface include materials having polyacrylic acid, polymethacrylic acid, styrenesulfonic acid, alginic acid, heparin, heparan sulfate, chondroitin sulfate, or dermatan bonded to the surface. Among these, a material having a carboxyl group on the surface is particularly preferable. Further, it is preferable that the surface of the material is smooth because it has excellent non-adhesiveness of cells.
[0009]
In the present invention, a method for forming a part or the whole of a container with a gas-permeable material includes a method for forming a part or the whole of a side wall of the container with a non-porous material having good gas permeability, and a method for forming a container with a gas-permeable material. There is a method in which a gas exchange is performed by attaching a porous membrane to the portion.
[0010]
Materials having good gas permeability include silicone resin, poly-4-methylpentene 1, polyisoprene, polybutadiene, ethylene-vinyl acetate copolymer, low-density polyethylene, and polystyrene. These materials have relatively good gas permeability among plastic materials, but since the gas permeability decreases as the thickness increases, it is usually preferably 200 μm or less, and preferably 100 μm or less. Particularly preferred. In the present invention, the entire container may be formed of such a material, or only a part thereof may be formed of such a material. The required gas permeability varies depending on the surface area of the container, the amount of packed cells, the type of cells, storage conditions, and the like, but it is necessary that the amount of cells filled in the container be sufficient to survive. .
[0011]
Other examples of the gas permeable material include a porous membrane. In the case of a porous membrane, the pore diameter needs to be smaller than a predetermined value in order to prevent the liquid inside the container from leaking. The preferred pore diameter is 1 μm or less, and particularly preferably 0.4 μm or less from the viewpoint of preventing leakage of liquid and preventing bacteria from entering the container. Examples of the material of the porous film include polytetrafluoroethylene, tetrafluoroethylene-hexafluoropropylene, polyethylene terephthalate, and polypropylene. These porous membranes may be used on the entire side wall of the container or on a part thereof. Another method of using a porous membrane is to attach the porous membrane to the container mouth. That is, if a cap provided with a porous membrane is fitted in the container mouth, gas exchange is performed in this portion, and the other portion of the container may be gas-impermeable.
[0012]
As described above, when the container of the present invention is provided with a function as a syringe, it is convenient that cells stored in the container can be directly injected into a living body. The syringe can have a structure similar to that of a general syringe for injecting medicines, etc., but if the cell-containing liquid stored inside can be pushed out, it has a different structure and principle from the conventional syringe. There may be. That is, a general syringe is composed of an outer cylinder, a syringe needle connection portion formed at the tip of the outer cylinder, and a piston. However, such a structure may be used, or the container may be made bellows or flexible. When extruding the cell-containing liquid inside the container, the container may be compressed or crushed.
[0013]
1A to 1C show an embodiment in which the container of the present invention has a function of a syringe. In this embodiment, the structure is the same as that of a conventional syringe for drug injection, and the outer cylinder side surface is formed of a gas permeable material. That is, the syringe includes an outer cylinder 1 and a piston 2, and a gasket 3 made of an elastic material is attached to a tip of the piston 2. The outer cylinder 1 is composed of a frame 4a made of a hard material and a cylindrical gas permeable sheet 4b adhered to the inner surface of the frame 4a. The frame 4a has a plurality of windows forming through holes. 5 are provided. Therefore, the gas permeable sheet is exposed to the outside at this window portion, and gas exchange is performed. Reference numeral 7 denotes a cap for sealing the injection needle connection portion 6. When injecting the cells inside the living body, the cap is removed and the injection needle is attached. In the embodiment of the window 5 in FIG. 1 shown in this embodiment, the shape of the window 5 is rectangular and the number of the windows 5 is four, but the present invention is limited to this embodiment only. It is not done. For example, the effects of the present invention can be achieved even in other embodiments with respect to the shape, dimensions, and number of the windows 5.
[0014]
FIG. 2 is a drawing showing a second embodiment of the present invention. In this example, the container 8 is formed in a bellows shape, and the injection needle connection portion 6 is provided at the tip thereof. The entire surface of the container 8 is formed of a gas permeable material, and high gas permeability can be achieved. In the case of this embodiment, the structure is simpler than that of the embodiment of FIG. In addition, there is no risk of liquid leakage from the piston seal during storage. When injecting the cells inside the living body, the rear end of 8 may be pressed directly with a finger or by using a jig.
[0015]
FIG. 3 shows a third embodiment of the present invention. In this embodiment, the cells are stored in a bag-shaped container 9 made of a flexible and gas-permeable material. The container 9 is provided with an injection needle connection portion 6. When injecting cells into a living body, the container 9 is housed in the outer cylinder 1, the injection needle is connected to 6, and then the container is connected with the piston 2. Press to push out the cells inside. In this example, the cells are pushed out by the action of the piston, so that the operation is easy. Since the cells are stored in the container, there is no need to worry about liquid leakage from the piston seal portion which is a problem in the embodiment shown in FIG. Further, the outer cylinder and the piston can be used repeatedly.
[0016]
FIG. 4 shows a fourth embodiment of the present invention. In this embodiment, the cells are stored in a flexible bag 10 as in the third embodiment, but the bag is not provided with a syringe needle connection portion. The bag is pressed against the bag breaking means 11 provided inside 1, the bag 10 is torn, and the cell-containing liquid inside flows out into the outer cylinder and is pushed out by the piston. The bag breaking means 11 is preferably one that can easily break the bag, such as a metal blade or a needle.
[0017]
FIG. 5 shows a fifth embodiment of the present invention. In this embodiment, cells are stored in a bag 12 made of a flexible material. An injection needle connection portion 6 is provided at one end of the container, and the cells are pushed out of the container by squeezing the container 12 with an ironing member 13.
[0018]
FIG. 6 shows an embodiment in which a gas permeable porous membrane is attached to the cap of the injection needle connecting portion to have a gas permeable function. As can be seen, in this embodiment, the container comprises an outer cylinder 1, a piston 2, a gasket 3 and a cap 12. Further, a hydrophobic gas-permeable membrane 14 is attached to the cap 7, and gas exchange is performed through the membrane 14.
[0019]
In the case where the container has the function of a syringe, it is preferable to adopt a structure in which the loss of cells is as small as possible when pushing out the cells inside. For that purpose, the shape of the container and the shape of the syringe connection may be devised. It is preferable that the residual cells can be reduced because the efficiency of cell utilization is increased.
[0020]
The sterilized container of the present invention is used for preserving cells. The cells are usually stored together with the medium, but injecting them directly into the living body requires the use of a medium that is safe for the living body. For example, when injecting chondrocytes or its precursor cells into patients with osteoarthritis, when injecting neurons or their precursor cells into the brain of patients with Parkinson's disease, and when injecting cardiomyocytes into patients with heart disease When cells are injected into humans, a synthetic medium containing no biological components such as bovine serum or a medium using the patient's own serum is preferred. There is no particular limitation on the cells to be housed therein, but stem cells cultured by cell engineering techniques or cells obtained by differentiating stem cells into target cells are preferable. Genetically modified cells obtained by modifying these cells by genetic engineering techniques are also preferred.
[0021]
The stem cells include embryonic stem cells (embryonic stem cells: ES cells), embryonic germ cells (embryonic germ cells: EG cells), and somatic stem cells (adult stem cells: adult stem cells; AS cells). Cell lines include bone cells, chondrocytes, muscle cells, cardiomyocytes, nerve cells, tendon cells, adipocytes, pancreatic cells, hepatocytes, skin (epidermal cells / fibroblasts), blood cells, etc. it can.
[0022]
Although the container of the present invention is used for storing cells, it may be used as a cell culture container, and then the cells may be stored as they are. If a scaffold is required for culturing, it is preferable to use microparticles made from a material to which cells can adhere. Among such materials, those formed of a bioabsorbable material are preferable because they are absorbed and do not remain after being injected into a living body. Examples of suitable materials include polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, lactic acid-caprolactone copolymer, trimethylene carbonate, polydioxanone, and collagen. Microparticles are preferred because they are porous and can adhere large amounts of cells.
[0023]
Cell culture can be performed by a known method. That is, cells are separated from a living body, and stem cells are selectively separated therefrom, and then cultured by adding a cell growth factor or a growth factor. The cultivation is preferably performed in an incubator. The cultured cells are stored in the container of the present invention and stored under conditions suitable for storage until needed for treatment. Storage is preferably performed at a low temperature, but for a short period of time, storage can be performed at room temperature or under heating.
[0024]
By injecting the cells contained in the container of the present invention into a living body or a vein, osteoarthritis, rheumatoid arthritis, pseudoarthritis, progressive muscular dystrophy, myocardial infarction, stroke, Parkinson's disease, spinal cord injury, tendon injury It is applied to the treatment of diabetes, liver dysfunction, digestive dysfunction, skin damage, leukemia, blood system disease and the like.
[0025]
【The invention's effect】
If the cells are stored using the container of the present invention, the operation of detaching the cells from the culture container and the operation of washing, which are required in the conventional method, become unnecessary. In addition, if the container has the function of a syringe, cells can be directly injected into a living body, so that regenerative medicine can be easily and safely performed even in a facility without facilities. Furthermore, culturing cells in the container of the present invention is the most efficient because culturing, preserving, and injecting a living body can be performed by simple operations. If at least a part of the container is formed of a gas-permeable material, there is an advantage that cells can be stored for a long time.
[Brief description of the drawings]
1A is an overall perspective view showing an embodiment in which a container of the present invention has a function of a syringe, FIG. 1B is a front view of the container of the present invention, and FIG. FIG. 2 is a sectional view taken along line XX ′. FIG. 2 is a front view showing a second embodiment of the present invention having a syringe function. FIG. 3 is a third embodiment of the present invention having a syringe function. FIG. 4 is a partial sectional front view showing an example. FIG. 4 is a partial sectional front view showing a fourth embodiment of the present invention having a function of a syringe. FIG. 5 is a fifth embodiment of the present invention having a function of a syringe. FIG. 6 is a front view showing an embodiment of the present invention in which a gas permeable material is provided at the injection needle connection part.
1. 1. syringe barrel Piston 3. Gasket 4a. Frame 4b. Gas permeable sheet5. Window6. Injection needle connection 7. Cap8. 8. bellows-shaped container Bag-shaped container 1
10. Bag-shaped container 2
11. Bag breaking means 12. Bag-shaped container 3
13. Ironing member 14. Porous membrane

Claims (35)

A cell storage container in which the inner surface of the container is formed of a material to which cells are unlikely to adhere. 2. The cell storage container according to claim 1, wherein the material to which cells are not easily adhered is a material having a hydrophilic material bonded or coated on the surface. Hydrophilic material, from acrylamide polymer, methacrylamide polymer, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, cellulose, dextran, hyaluronic acid, glycosumming glycan, proteoglycan, carrageenan and protein The cell storage container according to claim 2, which is a material selected from the group consisting of: The cell storage container according to claim 1, wherein the material to which cells are hardly adhered is a material having a negative charge on the surface. The cell storage container according to claim 4, wherein the material having a negative charge on the surface is a material having polyacrylic acid, polymethacrylic acid, styrenesulfonic acid, alginic acid, heparin, heparan sulfate, chondroitin sulfate or dermatan bound to the surface. 2. The cell storage container according to claim 1, wherein the material to which cells are not easily adhered is a material having a hydrophobic material bound or coated on the surface. 7. The cell storage container according to claim 6, wherein the hydrophobic material is a fluororesin or a silicone resin. The cell storage container according to claim 1, wherein the container has a function as a syringe. 9. The cell storage container according to claim 8, wherein the container is formed in a bellows shape, and an inner solution containing cells is extruded by compressing the bellows portion, thereby exhibiting a function as a syringe. 9. The cell storage container according to claim 8, wherein the container is formed of a flexible material, and the internal solution containing cells is extruded by crushing the flexible portion, thereby functioning as a syringe. 9. The cell storage container according to claim 8, wherein the container comprises a bag for storing cells, a syringe capable of storing the bag, and a bag breaking means provided in the syringe. 9. The cell storage container according to claim 8, wherein the container is formed of a flexible material, and the inner solution containing the cells is pushed out by squeezing the flexible portion, thereby exhibiting a function as a syringe. The cell storage container according to any one of claims 1 to 12, wherein a part or all of the container is formed of a gas-permeable material. 14. The cell storage container according to claim 13, wherein the gas permeable material is a non-porous sheet formed of a synthetic resin having good gas permeability. 15. The cell storage container according to claim 14, wherein the synthetic resin having good gas permeability is silicone resin, poly (4-methylpentene) 1, polyisoprene, polybutadiene, ethylene-vinyl acetate copolymer, low-density polyethylene, or polystyrene. 14. The cell storage container according to claim 13, wherein the gas permeable material is a porous membrane made of a hydrophobic material. 17. The cell storage container according to claim 16, wherein at least a part of the container side wall is formed of a porous membrane. 17. The cell storage container according to claim 16, wherein the porous membrane is attached to a cap attached to a container opening. The cell storage container according to any one of claims 16 to 18, wherein the porous membrane has a pore diameter of 1 µm or less. The cell storage container according to any one of claims 16 to 18, wherein the pore diameter of the porous membrane is 0.4 µm or less. The cell storage container according to any one of claims 16 to 20, wherein the hydrophobic material is polytetrafluoroethylene, tetrafluoroethylene-hexafluoropropylene copolymer, polyethylene terephthalate, or polypropylene. A cell product obtained by filling the container according to any one of claims 1 to 21 with a cell-containing liquid. 23. The cell product according to claim 22, wherein the cell product is therapeutic. 24. The cell product according to claim 22, wherein the cells are embryonic stem cells or somatic stem cells. 24. The cell product according to claim 22, wherein the cell is a bone cell. 24. The cell product according to claim 22, wherein the cells are chondrocytes. The cell product according to claim 22 or 23, wherein the cell is a muscle cell. The cell product according to claim 22 or 23, wherein the cell is a cardiomyocyte. 24. The cell product according to claim 22, wherein the cell is a nerve cell. The cell product according to claim 22 or 23, wherein the cell is a tendon cell. The cell product according to claim 22 or 23, wherein the cell is an adipocyte. 24. The cell product according to claim 22, wherein the cells are pancreatic cells. The cell product according to claim 22 or 23, wherein the cell is a hepatocyte. 24. The cell product according to claim 22, wherein the cells are skin cells. 24. The cell product according to claim 22, wherein the cell is a blood cell.

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