JP2007161307A - Refrigerating receptacle, its manufacturing method and refrigerating method of living specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a refrigerating receptacle in which liquid nitrogen and/or various kinds of minor germs are hardly allowed to enter the receptacle and the receptacle is suitable for preservation of a specimen of a rare living thing. <P>SOLUTION: This invention relates to a receptacle for refrigerating the specimen of a living thing. This receptacle has a receptacle substrate made of cold resistant plastic material provided with an opening part and a bottom part and a sealing part having an inner cavity and constituted by a cold resistant film. The sealing part has an upper end part protruded higher than the opening part, and the refrigerating receptacle is connected to the opening part of the receptacle substrate in such a way that the inner cavity communicates with the opening part. In the refrigerating receptacle, the specimen of the living thing stored in the receptacle is not contaminated because liquid nitrogen and/or various kinds of minor germs are hardly allowed to enter the receptacle. In addition, when defrosting work is carried out under a state in which liquid nitrogen is entered into the receptacle, liquid nitrogen is rapidly expanded, the receptacle is prevented from being broken and a worker does not have any opportunity suffering hurt. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cryopreservation container, a method for producing the same, and a cryopreservation method. Specifically, it is a container for cryopreserving a biological sample, which has a mouth portion and a bottom portion, a container base portion made of cold-resistant plastic, a lumen, and a seal made of a cold-resistant film. The sealing portion is cryopreserved in which the upper end portion of the sealing portion protrudes above the mouth portion and is coupled to the mouth portion of the container base so that the lumen and the mouth portion communicate with each other. Concerning the container.

  In recent years, the field of cell medicine such as regenerative medicine, cell-containing medicine and infertility treatment has made remarkable progress. In these cell medical fields, a technique for cryopreserving a biological sample is an indispensable technique to be carried out by all persons skilled in the art.

  As a container for cryopreserving cells, the present applicant has disclosed an invention of a bag formed by a laminated film of an ultrahigh molecular weight polyethylene layer and a low density polyethylene layer. This invention is an invention filed by the present applicant and has already been patent-registered (Patent Document 1). The product of the present invention has contributed to the development of the cord blood bank in Japan in recent years. In addition to the above-mentioned cryopreservation bag, a film made of a laminated film of a polyimide film and a fluorinated ethylene propylene polymer film (Patent Document 2), or a film made of a copolymer film of tetrafluoroethylene and ethylene (Patent Document) 3) etc. have been proposed. Furthermore, Patent Document 4 discloses a cryopreservation container formed of an ethylene-vinyl acetate copolymer film that has been irradiated with an electron beam and biaxially stretched. Patent Document 5 discloses a cryopreservation container formed of a biaxially stretched crosslinked polyethylene film.

  In the procedure of cryopreserving suspension cells using such a bag, after preparing a liquid in which cells are suspended, the suspension is injected from a port provided in the bag or a tube connected to the port. Next, it seals by welding the port with which the bag was equipped, or the tube couple | bonded with the port. Then, the bag containing the floating cells is immersed in liquid nitrogen.

  By the way, polypropylene cryovials are generally used for cryopreservation of biological samples at the laboratory level. The cryovial has a typical vial structure that is capped by screwing the vial base and the cap as shown in FIG. The cryovial is used by storing a biological sample in a container, covering with a cap, and immersing and storing in liquid nitrogen.

  However, when the cryovial containing the biological sample is stored in liquid nitrogen, liquid nitrogen and / or miscellaneous bacteria (mainly mycoplasma) present in liquid nitrogen from the gap between the container body and the cap of the cryovial are stored. There is a risk of entering the container. As a result, the biological sample in the container is contaminated by various germs (mainly mycoplasma) present in liquid nitrogen. Further, when the vial is taken out from the liquid nitrogen tank in a state where liquid nitrogen has entered the container, the liquid nitrogen rapidly expands due to a rise in the temperature in the container. As a result, the vial ruptures or the cap is fired. Then, an accident occurs in which the worker is injured by the ruptured container piece or the fired cap. If an operator is injured in such an accident and the biological sample in the container is contaminated with germs in liquid nitrogen, there is a risk that the germ will enter the wound of the worker and lead to a larger accident. is there.

As a means for solving such a problem, it is conceivable to use the bag described in Patent Documents 1 to 5. However, although the bags of Patent Documents 1 to 5 are suitable for preserving suspension cells such as red blood cells, white blood cells and hematopoietic stem cells, they are ES cells, mesenchymal stem cells, skeletal mononuclear cells, sperm cells, and eggs. For rare cells such as cells, islets, mucosal epithelial tissue, corneal epithelial tissue and cultured corneal tissue, and rare microorganisms such as microbial mutants and microorganisms that are difficult to collect, as well as sperm and egg storage It is not necessarily suitable. The following four points can be cited as the reason.
-When a preservation | save object is a structure | tissue, it is difficult to accommodate a structure | tissue in a bag aseptically in the bag provided with the port.
-When there are few sperm and eggs to be stored, it is difficult to specify the positions of sperm and eggs in the bag.
・ If the cells to be stored are adherent cells and microorganisms, the cells and microorganisms may adhere to the inner wall of the tube when the cells are accommodated in the bag from the tube connected to the port of the bag. The work of housing is difficult.
-Since the film which comprises a bag is flexible, there exists a possibility that the biological sample inside a bag may be damaged by the influence of an external force.

Japanese Patent No. 2876588 Japanese Patent Publication No.49-8079 Japanese Utility Model Publication No. 55-55069 Japanese Patent Publication No. 55-44977 Japanese Patent Publication No. 62-57351

  An object of the present invention is to provide a cryopreservation container suitable for preserving a rare biological sample, in which liquid nitrogen and / or bacteria are difficult to enter the container.

The present invention
[1] A container for cryopreserving a biological sample,
A container base comprising a mouth and a bottom and made of cold-resistant plastic;
Including a sealing portion comprising a lumen and made of a cold-resistant film;
The sealed portion has a cryopreservation container in which an upper end portion of the sealed portion protrudes above the mouth portion and is coupled to the mouth portion of the container base so that the lumen and the mouth portion communicate with each other.
[2] The cryopreservation container according to [1], wherein the cold-resistant plastic has a flexural modulus of 800 to 2000 MPa,
[3] The cryopreservation container according to [1], wherein the film thickness of the cold resistant film is 25 to 500 μm,
[4] The material of the cold resistant plastic is polypropylene,
Cold resistant film
Any one polymer layer selected from the group consisting of low density polyethylene, medium density polyethylene, ethylene-vinyl acetate copolymer, and ethylene-methacrylic acid ester;
The cryopreservation container according to [1], which is a polyethylene laminated film including a layer of ultrahigh molecular weight polyethylene,
[5] The material of the cold resistant plastic is polytetrafluoroethylene,
The cryopreservation container according to [1], wherein the cold resistant film is a fluoropolymer film containing at least a fluoropolymer layer,
[6] The cryopreservation container according to [5], wherein the fluorine-containing polymer film further includes a polyimide layer,
[7] A method for producing a container for cryopreserving a biological sample,
(I) a step of preparing a container base having a mouth portion and a bottom portion and made of cold-resistant plastic;
(II) preparing a cylindrical body having a lumen and made of a cold-resistant film; and
(III) The cylindrical body is sealed by being coupled to the mouth of the container base so that the upper end of the tubular body protrudes above the mouth and the lumen and the mouth communicate with each other. A method for producing a cryopreservation container including a step of forming a stopper,
[8] A method for producing a container for cryopreserving a biological sample,
(i) preparing a container base comprising a mouth and a bottom and made of cold-resistant plastic;
(ii) bonding at least one cold resistant film to the mouth of the container base such that a portion of the at least one cold resistant film protrudes above the mouth; and
(iii) The method for producing a cryopreservation container according to [7], including a step of forming a sealed portion by forming a lumen so that the combined cold-resistant film communicates with the mouth portion;
[9] A method of cryopreserving a biological sample in a cryopreservation container,
The cryopreservation container is:
A container base comprising a mouth and a bottom and made of cold-resistant plastic;
Including a sealing portion comprising a lumen and made of a cold-resistant film;
The sealing part is a container coupled to the mouth of the container base so that the upper end of the sealing part protrudes above the mouth and the lumen and the mouth communicate with each other.
The method
1) storing the biological sample in the container base;
2) a step of sealing the inside of the container base by welding the sealing portion; and 3) a method for cryopreserving a biological sample including a step of cryopreserving,
[10] The biological sample cryopreservation method according to [9], wherein the biological sample is an ES cell, a mesenchymal stem cell, a sperm cell, or an egg cell,
[11] The biological sample cryopreservation method according to [9], wherein the biological sample is sperm, ovum, pancreatic islet, mucosal epithelial tissue, corneal epithelial tissue, and cultured corneal tissue.

  According to the cryopreservation container of the present invention, a rare biological sample can be stored safely and easily. More specifically, in the cryopreservation container, since liquid nitrogen and / or various bacteria are difficult to enter the container, the biological sample stored in the container is not contaminated. Further, when a thawing operation or the like is performed in a state where liquid nitrogen has entered the container, the liquid nitrogen expands rapidly, preventing the container from being damaged, and there is no risk of injury to the operator. Furthermore, since the container base has a certain altitude, the biological sample is not damaged by an external force.

  The “cryopreservation container” in the present invention refers to a container that can store a biological sample, and is a container that is made of a material that can physically withstand an environment of at least 0 degrees or less. Furthermore, it refers to a container in which liquid nitrogen and / or miscellaneous bacteria present in liquid nitrogen do not easily enter the container during cryopreservation. The configuration of the cryopreservation container of the present invention includes a container base having a mouth portion and a bottom portion and made of a cold-resistant plastic, and a sealing portion having a lumen and made of a cold-resistant film. Is connected to the mouth of the container base so that the upper end of the sealing part protrudes above the mouth and the lumen and the mouth communicate with each other.

  The “biological sample” is not particularly limited as long as it is derived from a living body that can be cryopreserved. For example, blood such as whole blood, red blood cells, white blood cells, plasma, platelets and platelet-rich plasma, Blood components and blood-derived suspension cells, rare suspension cells such as bone skeleton mononuclear cells and hematopoietic stem cells, rare cells such as ES cells, mesenchymal stem cells, sperm cells and egg cells, islets, mucosal epithelial tissues, Examples include tissues such as corneal epithelial tissue and cultured corneal tissue, microorganisms that are difficult to collect, microorganisms including microbial mutants such as drug-resistant tuberculosis, and sperm and eggs. Among these biological samples, from the viewpoint of not damaging the contents, rare adherent cells, tissues and microorganisms are preferable, more preferably rare cells, sperm and ovum, particularly preferably sperm cells, ovum cells, sperm and It is an egg.

  The above “freezing storage” refers to storage in an environment at 0 ° C. or less, but from the viewpoint of storage over a long period of time, it is preferably −20 ° C. or less, more preferably −80 ° C. or less, particularly preferably −196 This means storage in an environment of liquid temperature (liquid nitrogen temperature). Examples of the cryopreservation method include a method using a commercially available deep freezer and the like, and a method using a cooling substance such as dry ice, liquid nitrogen, and liquid helium. Among these cryopreservation methods, liquid nitrogen is preferable from the viewpoint of storage over a long period of time.

  The “container base” refers to a general container shape having a mouth portion and a bottom portion and having no lid, and is composed of a cold-resistant plastic. In the present invention, the “mouth” includes an area within 10 mm from the upper end of the container base. The shape of the container base is not particularly limited as long as it does not deviate from the general shape of the container, and examples thereof include a columnar shape, a prismatic shape, a conical shape, and a pyramidal shape. Among these shapes, manufacturing is easy, the shape is similar to that of the cryovial, and a columnar shape is preferable from the viewpoint of adapting to accessories such as a cane for attaching the cryovial. Also, the internal volume of the container base is not particularly limited, but it is similar in shape to a cryovial that is used to those skilled in the art, and can be applied to accessories such as a cane for attaching the cryovial. , About 0.5 to 5.0 ml, preferably about 1.0 to 2.0 ml, but is not limited thereto.

  The “cold resistant plastic” in the present invention has such a hardness that the container base is not easily deformed by an external force, and is at least 0 degrees or less, preferably −40 degrees or less, particularly preferably −196 degrees (liquid nitrogen temperature). It can be physically tolerated in the environment. Examples of the material for the cold resistant plastic include polypropylene, polycarbonate, and polytetrafluoroethylene. Further, the hardness of the cold resistant plastic is not particularly limited as long as the container base is not damaged, but the flexural modulus is about 900 MPa or more, preferably about 1000 to 2000 MPa, particularly preferably about 1200 to 1700 MPa. is there. Further, the thickness of the container base is not particularly limited as long as it has the above-mentioned breaking strength and does not inhibit the heat conduction in the cryopreservation. For example, when the material of the cold resistant plastic is polypropylene, the thickness of the container base is about 0.5 to 1.5 mm, preferably about 0.8 to 1.2 mm. When the material of the cold resistant plastic is polytetrafluoroethylene, the thickness of the container base is about 0.5 to 1.5 mm, preferably about 0.8 to 1.2 mm. Since the shape of these container bases can be appropriately set by those skilled in the art depending on the cold-resistant plastic to be selected, the present invention is not limited to these descriptions.

  A sealing portion made of a cold resistant film is provided at the mouth of the container base. The “sealing part” in the present invention is formed by welding to the mouth of the container base, and can seal the inside of the container base by welding, and is composed of a cold-resistant film. Say. That is, the sealing part of this invention plays the role of the lid | cover in a cryopreservation container. The sealing portion is formed by joining the mouth portion of the container base so that the upper end portion of the sealing portion protrudes above the mouth portion and the lumen and the mouth portion communicate with each other. Here, “bonding” in the present invention refers to a state where the container base and the sealing part are in contact with each other to an extent that separation between the container base and the sealing part is not easy due to adhesion, welding, embedding, and the like. The wall surface to be joined may be any of the inner wall of the mouth portion, the inside of the wall, and the outer wall, but the outer wall is preferable from the viewpoint of easy production, and the inside of the wall is preferred from the viewpoint of the bonding strength between the container base and the sealing portion. preferable.

The shape of the sealing part is, for example,
(A) A shape in which a lumen is formed by stacking two rectangular films of the same shape and welding two parallel sides of the two overlapping films (FIG. 1); and
(B) A shape in which a lumen is formed by overlapping two parallel sides of a rectangular film and welding the two overlapping sides (Fig. 2)
Etc. Among these shapes, from the viewpoint of easy welding when sealing, (A) two rectangular films of the same shape are overlapped, and the two parallel sides of the two overlapped films are welded to form a lumen. Although the formed shape is preferable, the shape of the sealing portion of the present invention is not limited to these descriptions.

  The “cold-resistant film” can be heat-welded with the cold-resistant plastic, has flexibility, is at least 0 ° or less, preferably −40 ° or less, particularly preferably −196 ° (liquid nitrogen temperature) ) Including a cold-resistant polymer layer that can physically withstand the environment. The film thickness of the cold resistant film is not particularly limited as long as it can be heat-welded and has flexibility, but it is, for example, about 25 to 500 μm, preferably about 50 to 300 μm, Especially preferably, it is about 75-200 micrometers.

  The structure and material of the cold resistant film must be a structure and material that can be thermally welded to the cold resistant plastic, and therefore depends on the material of the cold resistant plastic. For example, when the material of the cold resistant plastic is polypropylene, a polyethylene-based laminated film is mainly selected as the cold resistant film. For example, when the material of the cold resistant plastic is polytetrafluoroethylene, a fluorine-containing polymer film is mainly selected as the cold resistant film. The above two combinations can be appropriately selected by those skilled in the art, and are not particularly limited. This is because the former is preferable to the latter in terms of manufacturing cost, but the latter is superior to the former in terms of easy manufacturing.

  The “polyethylene-based laminated film” in the present invention is any one polymer layer selected from the group consisting of low-density polyethylene, medium-density polyethylene, ethylene-vinyl acetate copolymer, and ethylene-methacrylic acid ester, A laminated film including a layer of ultrahigh molecular weight polyethylene. That is, any one polymer layer selected from the group consisting of low-density polyethylene, medium-density polyethylene, ethylene-vinyl acetate copolymer, and ethylene-methacrylic acid ester serves to weld to the polypropylene, and has an ultra high molecular weight. Polyethylene imparts cold resistance to the laminated film. Among these polyethylene-based laminated films, a laminated film including a low-density polyethylene layer and an ultrahigh-molecular-weight polyethylene layer is preferable from the viewpoint of weldability with polypropylene and easy production of the film laminated film.

The “low density polyethylene” in the present invention refers to a general low density polyethylene in the field of polymer chemistry, and the density is about 0.925 g / cm ³ or less, preferably 0.910 to 0.925 g / cm. ³ polyethylene. Further, “medium density polyethylene” refers to a general medium density polyethylene in the field of polymer chemistry, and refers to a polyethylene having a density of 0.926 to 0.940 g / cm ³ .

  The method for producing any one polymer layer selected from the group consisting of low density polyethylene, medium density polyethylene, ethylene-vinyl acetate copolymer, and ethylene-methacrylate ester has a molecular structure, a glass transition temperature, and a melting point. Those skilled in the art can appropriately select and manufacture them. Examples include compression molding, injection molding, extrusion molding, T-die molding, inflation molding, and solvent casting.

  The “ultra high molecular weight polyethylene” means a polyethylene that has at least cold resistance and satisfies a molecular weight condition of one million or more by the viscosity method or three million or more by the light scattering method. Say. The upper limit of the molecular weight of ultrahigh molecular weight polyethylene is not particularly limited, but at present, about 6 million (viscosity method) is said to be the limit that can be produced by those skilled in the art. The ultra high molecular weight ethylene layer can be produced mainly by cutting, but the present invention is not limited thereto. Ultra high molecular weight polyethylene can be purchased from Sakushin Kogyo.

  Examples of the method for producing the polyethylene-based laminated film include a thermal laminating method, a thermal compression method, a high-frequency heating method, and a solvent casting method. Among these laminating methods, the thermal laminating method is preferable from the viewpoint of easy production. By this lamination, the two layers are firmly welded and the two layers are no longer peeled off. Here, the temperature condition in the thermal lamination method is, for example, about 150 when the polyethylene-based laminated film is a laminated film including a low density polyethylene layer and an ultrahigh molecular weight polyethylene layer from the viewpoint of safety in production. Although it is -250 degreeC, Preferably it is about 170-200 degreeC, This invention is not limited to this.

  The “fluorinated polymer film” refers to a film including a polymer layer (hereinafter referred to as a fluorine-containing polymer) having at least one fluorine atom in the main chain and / or side chain. Examples of the fluorine-containing polymer include tetrafluoroethylene, polyvinylidene fluoride, ethylene-tetrafluoroethylene copolymer, and tetrafluoroethylene-hexafluoropentene copolymer. In addition, a polymer obtained by introducing at least one fluorine atom into the main chain and / or side chain of a polymer by a method of fluorinating a general-purpose olefin-based polymer by plasma treatment using a fluorine gas atmosphere. It may be. A typical example is a fluorinated ethylene-propylene polymer (hereinafter also referred to as a fluorinated ethylene-propylene polymer). Among these fluorine-containing polymers, ethylene-tetrafluoroethylene copolymer and fluorinated ethylene-propylene polymer are preferable from the viewpoint of moldability and flexibility of the fluorine-containing polymer layer. Is not limited to this. Examples of commercially available fluorine-containing polymers include Nephron EFEP (trademark) manufactured by Daikin Industries.

  From the viewpoint of molding processability, the molecular weight of the fluorine-containing polymer is about 1,000 to 1,000,000, preferably about 2,000 to 500,000, and more preferably about 5,000 to 1,000 in terms of number average molecular weight. Although it is 300,000, it is not limited to this.

  Examples of the method for forming the fluoropolymer layer include compression molding, injection molding, extrusion molding, T-die molding, roll molding, inflation molding, and solvent casting. T-die molding is used from the viewpoint of molding processability. Roll molding and inflation molding are preferred, but the present invention is not limited to these.

  In addition, from the viewpoint of improving cold resistance, the fluorine-containing polymer film may be in the form of a laminated film in which a polymer layer other than the fluorine-containing polymer is further laminated on the fluorine-containing polymer layer. Examples of such a polymer include ultrahigh molecular weight polyethylene, polyimide, and ethylene-vinyl acetate copolymer, and polyimide is preferable from the viewpoint of thermal conductivity to the contents and cold resistance. In the present invention, such a laminated film is referred to as a “fluorinated polymer type“ laminated ”film”.

  The above “polyimide” refers to a heat-resistant polymer having an imide bond in the main chain, for example, a non-thermoplastic polyimide having only an imide bond in the main chain, a wholly aromatic polyimide, an organic solvent-soluble polyimide, a polyetherimide. And polyimide amide. The molecular weight of the polyimide is about 1,000 to 1,000,000, preferably about 2,000 to 500,000, more preferably about 5,000 to 300,000 in terms of number average molecular weight from the viewpoint of moldability. Although it is 000, it is not limited to this.

  Examples of the polyimide layer include those formed by a hot melting method, an extrusion method or a compression method, a solvent casting method, and the like at a high temperature and a high pressure.

  Examples of the method for laminating the fluorine-containing polymer layer on the polyimide layer include a thermal laminating method, a thermal compression method, a high-frequency heating method, and a solvent casting method. Among these laminating methods, the thermal laminating method is preferable from the viewpoint of ease of production. Here, the temperature condition in the thermal lamination method is about 200 to 300 ° C., preferably about 200 to 250 ° C., from the viewpoint of safety in manufacturing.

  In the container base and the sealing part of the present invention described above, the upper part of the sealing part protrudes above the mouth part, and the mouth part of the container base part communicates with the lumen and the mouth part. The cryopreservation container of this invention can be manufactured by couple | bonding with.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, but the present invention is not limited to these embodiments.

  FIG. 1 is one embodiment of the cryopreservation container of the present invention. The cryopreservation container of FIG. 1 includes a sealing container 2 having a mouth 11 and a bottom 12 and a container base 1 made of polypropylene and a lumen 21 made of two polyethylene laminated films. The sealing part 2 is coupled to the outer wall of the mouth part 11 of the container base so that the upper end part protrudes above the mouth part 11 and the lumen 21 and the mouth part 11 communicate with each other. Here, the polyethylene-based laminated film is a laminated film having an ultrahigh molecular weight polyethylene layer as an intermediate layer, and a low-density polyethylene layer on both sides of the ultrahigh molecular weight polyethylene layer. The container base 1 and the sealing portion 2 are joined by heat welding, and a welding margin 31 is formed around the mouth. Furthermore, since the sealing part 2 is comprised by two polyethylene-type laminated | multilayer film, two parallel sides are heat-welded and the welding margin 32 is formed. Accordingly, the horizontal cross section of the sealing portion 2 is shaped like a human eye.

  FIG. 2 is an embodiment of the cryopreservation container of the present invention, which is different from FIG. The cryopreservation container of FIG. 2 includes a sealing part 2 having a mouth part 11 and a bottom part 12 and a container base 1 made of polytetrafluoroethylene and a lumen 21 made of a single fluoropolymer laminate film. Including. The sealing part 2 is coupled to the inner wall of the mouth part 11 of the container base so that the upper end of the sealing part 2 protrudes above the mouth part 11 and the lumen 21 and the mouth part 11 communicate with each other. Here, the fluorine-containing polymer film is a two-layer film composed of a fluorinated ethylene-propylene copolymer layer and a polyimide layer. The container base 1 and the sealing portion 2 are bonded by thermal welding of polytetrafluoroethylene and a fluorine-containing polymer, and a welding margin 31 is formed around the mouth. Furthermore, since the sealing part 2 is composed of a single fluorine-containing polymer-based laminated film, the two overlapping parallel sides are heat-welded, and a welding margin 32 is formed. Accordingly, the horizontal section of the sealing portion 2 is circular.

  FIG. 3 shows an embodiment of the cryopreservation container of the present invention, which is different from FIGS. 1 and 2. The cryopreservation container of FIG. 3 includes a sealing container 2 having a mouth 11 and a bottom 12 and a container base 1 made of polypropylene and a lumen 21 made of a single polyethylene-based laminated film. The sealing part 2 is coupled to the inner wall of the mouth part 11 of the container base so that the upper end of the sealing part 2 protrudes above the mouth part 11 and the lumen 21 and the mouth part 11 communicate with each other. Here, the polyethylene-based laminated film is a laminated film having an ultrahigh molecular weight polyethylene layer as an intermediate layer, and a low-density polyethylene layer on both sides of the ultrahigh molecular weight polyethylene layer. The container base 1 and the sealing portion 2 are joined by heat welding, and a welding margin 31 is formed around the mouth portion 11. Furthermore, since the sealing part 2 is comprised by one polyethylene-type laminated | multilayer film, the two overlapping parallel sides are heat-welded and the welding margin 32 is formed. Accordingly, the horizontal section of the sealing portion 2 is circular.

  FIG. 4 shows an embodiment of the cryopreservation container of the present invention different from those shown in FIGS. The cryopreservation container of FIG. 4 includes a sealing container 2 having a mouth 11 and a bottom 12 and a container base 1 made of polypropylene and a lumen 21 made of a single polyethylene-based laminated film. The sealing part 2 is embedded in the wall of the mouth part 11 of the container base so that the upper end part protrudes above the mouth part 11 and the lumen and the mouth part communicate with each other. Here, the polyethylene-based laminated film is a laminated film having an ultrahigh molecular weight polyethylene layer as an intermediate layer, and a low-density polyethylene layer on both sides of the ultrahigh molecular weight polyethylene layer. The container base 1 and the sealing part 2 are bonded to each other in a mold having an outer diameter slightly larger than that of the container shown in FIG. Thereafter, at least the welding margin 31 is filled with a melt of the same material (polypropylene resin) as that of the container base 1, so that the sealing part 2 is embedded in the wall of the mouth 11 of the container base and cooled to become the main part. The cryopreservation container of the invention is manufactured. Accordingly, the horizontal section of the sealing portion 2 is circular.

  FIG. 5 shows an embodiment of the cryopreservation container of the present invention different from those shown in FIGS. The cryopreservation container of FIG. 5 includes a sealing container 2 having a mouth 11 and a bottom 12 and a container base 1 made of polypropylene and a lumen 21 made of a single polyethylene-based laminated film. As the container base 1, a vial base of a commercially available cryovial as shown in FIG. 6 is used as it is. The sealing part 2 is coupled to the outer wall of the mouth part 11 of the container base so that the upper end part protrudes above the mouth part 11 and the lumen 21 and the mouth part 11 communicate with each other. Here, the polyethylene-based laminated film is a laminated film having an ultrahigh molecular weight polyethylene layer as an intermediate layer, and a low-density polyethylene layer on both sides of the ultrahigh molecular weight polyethylene layer. The container base 1 and the sealing portion 2 are joined by heat welding of polypropylene and a polyethylene-based laminated film, and a welding margin 31 is formed around the mouth portion 11. Furthermore, since the sealing part 2 is comprised by one polyethylene-type laminated | multilayer film, the two overlapping parallel sides are heat-welded and the welding margin 32 is formed. Accordingly, the horizontal section of the sealing portion 2 is circular. Furthermore, since the container base 1 is a vial base of a commercially available cryovial, the production of the cryopreservation container of the present invention is facilitated. In addition, since the commercially available cryovial further includes a cap 4 that is screwed into the vial base, it is convenient in handling after freezing and thawing, and contamination can be prevented.

  Hereinafter, the method for producing a cryopreservation container of the present invention will be described with reference to preferred embodiments, but the present invention is not limited to these preferred embodiments.

The present invention also includes a method for producing the cryopreservation container. The first method for producing a cryopreservation container in the present invention includes the following steps (I) to (III).
(I) a step of preparing a container base having a mouth portion and a bottom portion and made of cold-resistant plastic;
(II) preparing a cylindrical body having a lumen and made of a cold-resistant film; and
(III) The cylindrical body is sealed by being coupled to the mouth of the container base so that the upper end of the tubular body protrudes above the mouth and the lumen and the mouth communicate with each other. Forming the stop.

  That is, the above first manufacturing method includes the steps of (I) preparing the container base, (II) preparing the cylindrical body, and (III) coupling the cylindrical body to the container base. The method for producing the cryopreservation container of the present invention.

  The above "(I) Step of preparing a container base having a mouth part and a bottom part and made of cold-resistant plastic" means manufacturing a container having a general lid-like shape made of cold-resistant plastic. Or it refers to a process that exists at the manufacturer's hand by purchase. Examples of the method for producing the container base include compression molding, injection molding, extrusion molding, and vacuum molding. Moreover, you may use the vial base part of a commercially available cryovial as it is.

  The above-mentioned “(II) Step of preparing a cylindrical body having a lumen and made of a cold-resistant film” means that a cylindrical body made of a cold-resistant film is manufactured or purchased separately from the container base. The process of making it in the state which exists in a manufacturer's hand by doing. Here, the “cylindrical body” refers to a precursor having a lumen and capable of forming a sealing portion. As a method of manufacturing this cylindrical body, a heat welding method is mainly used. The welding width of the thermal welding method in the production of the cylindrical body is about 2 to 20 mm, preferably about 5 to 15 mm from the viewpoint of seal strength, but is not limited thereto. Further, the welding temperature can be appropriately designed by those skilled in the art depending on the melting point of the material to be used, and is not particularly limited. For example, when the cold resistant film is a laminated film of a polyethylene layer and an ultrahigh molecular weight polyethylene layer, the temperature is about 150 to 250 ° C., preferably about 170 to 200 ° C. from the viewpoint of sealing strength. For example, when the cold resistant film is a laminated film of a polyimide layer and a fluorine-containing polymer layer, the temperature is about 200 to 350 ° C., preferably about 200 to 300 ° C., from the viewpoint of sealing strength.

  And the cryopreservation container of this invention is completed by couple | bonding the cylindrical body prepared at the process of (II) to the container base prepared at the process of (I). More specifically, the cylindrical body is coupled to the mouth portion of the container base so that the upper end portion of the tubular body protrudes above the mouth portion and the lumen and the mouth portion communicate with each other. To form a sealing portion. Examples of the bonding method include adhesion with an adhesive and thermal welding, but the thermal welding method is preferable from the viewpoint of cold resistance of the cryopreservation container. The welding width at the time of joining the cylindrical body to the container base by the thermal welding method is about 2 to 20 mm, preferably about 5 to 15 mm from the viewpoint of sealing strength, but is not limited thereto. . Moreover, the temperature conditions in the heat welding method can be appropriately set by those skilled in the art depending on the combination of the cold resistant plastic and the cold resistant film. For example, when the cold resistant plastic is polypropylene and the cold resistant film is a laminated film of a polyethylene layer and an ultrahigh molecular weight polyethylene layer, it is about 150 to 250 ° C., preferably about 170 to 200 ° C. from the viewpoint of sealing strength. It is. Further, for example, when the cold resistant plastic is polytetrafluoroethylene and the cold resistant film is a laminated film of a polyimide layer and a fluorine-containing polymer layer, about 200 to 350 ° C. from the viewpoint of sealing strength, preferably About 200-300 degreeC.

The second method for producing a cryopreservation container in the present invention includes the following steps (i) to (iii).
(i) preparing a container base comprising a mouth and a bottom and made of cold-resistant plastic;
(ii) coupling at least one cold resistant film to the mouth of the container base such that a portion of the at least one cold resistant film protrudes above the mouth; and
(iii) A step of forming a sealed portion by forming a lumen so that the combined cold-resistant film communicates with the mouth portion.

  That is, the above-mentioned second manufacturing method includes the steps of (i) a step of preparing a container base, (ii) a step of bonding a cold-resistant film to the container base, and (iii) a step of forming a sealing portion. The method for producing the cryopreservation container of the present invention.

  The first manufacturing method described above is a method in which the inner cavity (cylindrical body) of the sealing portion is formed in advance and then bonded to the container base, whereas the second manufacturing method is a cold resistant film. Is connected to the container base, and then a lumen is formed to form a sealing portion. Therefore, the manufacturing method in (ii) the step of bonding the cold resistant film to the container base and (iii) the step of forming the sealing portion is achieved mainly by thermal welding. Furthermore, the conditions for heat welding may be the same as those in the first manufacturing method.

The present invention further extends to a method for cryopreserving biological samples using the cryopreservation container of the present invention. More specifically, the following steps 1) and 2) are included.
1) storing the biological sample in the container base;
2) a step of sealing the inside of the container base by welding the sealing portion; and 3) a step of cryopreserving.

  The “1) step of storing the biological sample in the container base” means a step of placing the biological sample in the container base, and a step of sealing the biological sample so that the biological sample can be sealed. Say. A method for storing the biological sample in the container base can be appropriately selected by those skilled in the art depending on the type of biological sample to be stored.

For example, when preserving cells such as egg cells, sperm cells, ES cells, or microorganisms such as drug-resistant M. tuberculosis, the cell suspension is obtained by suspending the cells in a medium or cell preservation solution that enables cryopreservation. Can be accommodated in the container base. Here, as a technique for accommodating, for example, a method disclosed in Japanese Patent Application Laid-Open No. 2000-189155 or a method of accommodating using the device shown in FIG. 7 (international patent application number PCT / JP2005 / 021962). Can be mentioned. In particular, it is preferable to use the device shown in FIG. 7 in that rare cells are not lost and the tissue can be cryopreserved without being damaged. Specifically, after the droplet of the cell suspension is dropped on the device shown in FIG. 7, it may be stored in the cryopreservation container of the present invention so that the droplet does not fall from the device. The cell concentration in the cell suspension can be appropriately determined by those skilled in the art depending on the cell type, and is not particularly limited. For example, when the cell is a sperm cell, the concentration is about 2 × 10 ^{7 to} 20 × 10. ⁷ cells / ml, or 1-10 cells / ml for oocyte cells. In addition, for example, about 10% dimethyl sulfoxide (DMSO) and 5 to 10% albumin may be added to the cell suspension as frost damage protection components.

  On the other hand, for example, when storing a tissue, the tissue can be infiltrated with, for example, physiological saline and stored in the container base. Here, as a technique for accommodating, for example, a method disclosed in Japanese Patent Application Laid-Open No. 2003-009845 or a method of accommodating using the device shown in FIG. In particular, it is preferable to use the device shown in FIG. 3 in that it can be stored frozen without damaging the tissue. Specifically, after placing or winding the tissue on the device shown in FIG. 7, the tissue may be stored in the cryopreservation container of the present invention so that the tissue does not fall off the device.

  Next, 2) the inside of the container base is sealed by welding the sealing portion. Here, “sealing” means that, for example, when a biological sample is stored under liquid nitrogen, liquid nitrogen and bacteria (mainly mycobacteria) present in liquid nitrogen do not enter the container base. . For the welding, for example, thermal welding is performed using an impulse sealer or the like. The temperature of welding can be appropriately set by those skilled in the art depending on the type of material of the cold resistant film. For example, when the cold resistant film is a laminated film of a polyethylene layer and an ultrahigh molecular weight polyethylene layer, the temperature is about 150 to 250 ° C., preferably about 170 to 200 ° C. from the viewpoint of sealing strength. For example, when the cold resistant film is a laminated film of a polyimide layer and a fluorine-containing polymer layer, the temperature is about 200 to 350 ° C., preferably about 200 to 300 ° C., from the viewpoint of sealing strength. By these welding operations, for example, the appearance of the cryopreservation container as shown in FIG. 1 becomes as shown in FIG.

  The biological sample sealed in the container base by the above steps is stored frozen. As described above, the temperature condition at the time of cryopreservation refers to storage in an environment at 0 ° C. or less, but from the viewpoint of storage over a long period, it is preferably −20 ° C., more preferably −80 ° C., particularly It is preferably in an environment of -196 degrees (liquid nitrogen temperature). Examples of the cryopreservation method include a method using a commercially available device such as a deep freezer, and a method using a cooling substance such as dry ice, liquid nitrogen, and liquid helium. Among these cryopreservation methods, liquid nitrogen is preferable from the viewpoint of storage over a long period of time.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

<Example 1>
The cryopreservation container of FIG. 1 was manufactured. Specifically, first, a cylindrical container base 1 having a mouth portion 11 and a bottom portion 12 was manufactured by injection molding polypropylene. The melting temperature in injection molding was about 210 degrees. The outer diameter of the mouth 11 of the container base 1 was about 10 mm, the height was 40 mm, and the inner volume of the container base 1 was 2.0 ml. Moreover, the bending elastic modulus of the polypropylene at this time was about 1500 MPa.

  Next, a laminated film was produced by a thermal welding method in which an ultrahigh molecular weight polyethylene layer was provided as an intermediate layer and a low density polyethylene layer was provided on both sides of the ultrahigh molecular weight polyethylene layer. Specifically, a linear low-density polyethylene layer (Idemitsu Chemical Co., Ltd.) having a thickness of about 50 μm on both sides of an ultra-high molecular weight polyethylene layer (weight average molecular weight of about 5.5 million, made by Sakushin Chemical Industry Co., Ltd.) having a thickness of about 75 μm. A three-layer polyethylene-based laminated film was produced. That is, the total film thickness of the polyethylene-based laminated film was about 175 μm. The temperature for heat welding was about 170 degrees. Then, two films prepared by cutting the polyethylene-based laminated film into a rectangular shape having a short side of 25 × long side of 30 mm were prepared. The two films were overlapped, and the long side and the two sides were heat-welded with a welding width of 5 mm, whereby a cylindrical body having a lumen 21 was manufactured.

  The mouth portion 11 of the container base 1 was inserted by about 10 mm into the lumen 21 of the cylindrical body. That is, the cylindrical body is arranged around the outer wall of the mouth 1 of the container base 1. A cylindrical body located around the outer wall of the mouth 1 of the container base 1 was thermally welded to the mouth 11 of the container base 1. The welding temperature at this time was about 170 degrees. The cryopreservation container shown in FIG. 1 of the present invention was manufactured by the above manufacturing method.

<Example 2>
The cryopreservation container shown in FIG. 2 was produced. Specifically, first, a cylindrical container base 1 having a mouth portion 11 and a bottom portion 12 was manufactured by injection molding of polytetrafluoroethylene. The melting temperature in injection molding was about 300 degrees. The outer diameter of the mouth 11 of the container base 1 was about 10 mm, the height was 40 mm, and the inner volume of the container base 1 was 2.0 ml. Moreover, the bending elastic modulus of the polytetrafluoroethylene at this time was about 1800 MPa.

  Next, one fluoropolymer-based laminated film (provided by Daikin Industries, film thickness of about 100 μm, short side 35 × long side 40 mm) was prepared.

  The short side of the fluorine-containing polymer-based laminated film was wound around the outer wall of the mouth portion 11 of the container base 1 so as to overlap 10 mm. A fluorine-containing polymer-based laminated film located around the outer wall of the mouth 1 of the container base 1 was thermally welded to the mouth 11 of the container base 1. Furthermore, the sealing part 2 provided with the lumen 21 was formed by heat-welding the long sides of the fluorine-containing polymer-based laminated film that had been overlapped by winding. The welding temperature at this time was about 190 degrees. The cryopreservation container shown in FIG. 2 of the present invention was manufactured by the above manufacturing method.

<Comparative Example 1>
A commercially available polypropylene cryovial was used. This cryovial is a general vial provided with a cap screwed into the container body as shown in FIG.

<Experimental example 1>
Next, a freezing test was performed using the cryopreservation container manufactured in Examples 1 and 2 and the cryopreservation container of Comparative Example 1. Specifically, 2.0 ml of an aqueous solution of 5% by volume of dimethyl sulfoxide (DMSO) was poured into the cryopreservation containers of Examples 1 and 2, and the inside was sealed by thermally welding the sealing portion. The thermal welding temperature at this time was about 170 degrees. On the other hand, the cryovial of Comparative Example 1 was capped by screwing a cap. Thereafter, these cryopreservation containers were immersed in liquid nitrogen and cryopreserved for 3 days. Thereafter, the cryopreservation container was taken out and thawed in a warm bath at about 37 to 40 ° C. These series of operations were performed 5 times each, and the state of the cryopreservation container at each operation was visually observed.

  As a result, the cap fired during the thawing operation of one example of Comparative Example 1. In this experimental example, care was taken not to point the cap toward the experimenter in all operations, so the accident was avoided, but the cryopreservation container of Comparative Example 1 might have liquid nitrogen intruding into the container. It was suggested. That is, the results showed the usefulness of the present invention.

  According to the cryopreservation container of the present invention, a rare biological sample can be stored safely and easily. More specifically, in the cryopreservation container, since liquid nitrogen and / or various bacteria are difficult to enter the container, the biological sample stored in the container is not contaminated. Further, when a thawing operation or the like is performed in a state where liquid nitrogen has entered the inside of the container, the liquid nitrogen rapidly expands to prevent the container from being damaged and the operator is not injured. Further, since the container base has a certain level of altitude, the biological sample is not damaged by an external force. Therefore, it can be used in banks for storing microorganisms or cells, for example, the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.

It is a figure which shows one embodiment of this invention. It is a figure which shows one embodiment different from FIG. 1 of this invention. FIG. 3 shows an embodiment different from FIGS. 1 and 2 of the present invention. It is a figure which shows one embodiment different from FIGS. 1-3 of this invention. It is a figure which shows one embodiment different from FIGS. 1-4 of this invention. It is a figure which shows the conventional cryopreservation container. FIG. 3 shows a device that can be used in the cryopreservation container of the present invention. It is a figure which shows the external appearance of the cryopreservation container which sealed the sealing part of the cryopreservation container of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container base 11 Mouth part 12 Bottom part 2 Sealing part 21 Lumen 31 Welding margin 32 formed when the container base part and the sealing part are joined 32 Welding margin 33 formed when the sealing part is formed Cryopreservation container Welding allowance 4 cap 41 screwing groove 5 device formed at the time of sealing

Claims (11)

A container for cryopreserving a biological sample,
A container base comprising a mouth and a bottom and made of cold-resistant plastic;
Including a sealing portion comprising a lumen and made of a cold-resistant film;
The sealed portion is a cryopreservation container in which an upper end portion of the sealed portion protrudes above the mouth portion and is coupled to the mouth portion of the container base so that the lumen and the mouth portion communicate with each other.   The cryopreservation container according to claim 1, wherein the cold-resistant plastic has a flexural modulus of 800 to 2000 MPa.   The cryopreservation container according to claim 1, wherein the film thickness of the cold resistant film is 25 to 500 µm. The material of the cold resistant plastic is polypropylene,
Cold resistant film
Any one polymer layer selected from the group consisting of low density polyethylene, medium density polyethylene, ethylene-vinyl acetate copolymer, and ethylene-methacrylic acid ester;
The cryopreservation container according to claim 1, wherein the cryopreservation container is a polyethylene laminated film including a layer of ultrahigh molecular weight polyethylene. The material of the cold resistant plastic is polytetrafluoroethylene,
The cryopreservation container according to claim 1, wherein the cold-resistant film is a fluorine-containing polymer film containing at least a fluorine-containing polymer layer.   The cryopreservation container according to claim 5, wherein the fluorine-containing polymer film further comprises a polyimide layer. A method for producing a container for cryopreserving a biological sample, comprising:
(I) a step of preparing a container base having a mouth portion and a bottom portion and made of cold-resistant plastic;
(II) preparing a cylindrical body having a lumen and made of a cold-resistant film; and
(III) The cylindrical body is sealed by being coupled to the mouth of the container base so that the upper end of the tubular body protrudes above the mouth and the lumen and the mouth communicate with each other. A method for producing a cryopreservation container comprising a step of forming a stopper. A method for producing a container for cryopreserving a biological sample, comprising:
(i) preparing a container base comprising a mouth and a bottom and made of cold-resistant plastic;
(ii) bonding at least one cold resistant film to the mouth of the container base such that a portion of the at least one cold resistant film protrudes above the mouth; and
(iii) The method for producing a cryopreservation container according to claim 7, comprising a step of forming a sealed portion by forming a lumen so that the combined cold-resistant film communicates with the mouth portion. A method for cryopreserving a biological sample in a cryopreservation container,
The cryopreservation container is:
A container base comprising a mouth and a bottom and made of cold-resistant plastic;
Including a sealing portion comprising a lumen and made of a cold-resistant film;
The sealing part is a container coupled to the mouth of the container base so that the upper end of the sealing part protrudes above the mouth and the lumen and the mouth communicate with each other.
The method
1) storing the biological sample in the container base;
2) A method for cryopreserving a biological sample, comprising: sealing the inside of the container base by welding the sealing portion; and 3) cryopreserving.   The method for cryopreserving a biological sample according to claim 9, wherein the biological sample is an ES cell, a mesenchymal stem cell, a sperm cell, or an egg cell. The method for cryopreserving a biological sample according to claim 9, wherein the biological sample is sperm, ovum, pancreatic islet, mucosal epithelial tissue, corneal epithelial tissue, and cultured corneal tissue.

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