JP2007302567A - Container for freeze preservation and method of freeze preservation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a container for freeze preservation without getting the invasion of liquid nitrogen, sundry bacteria or viruses into the inside of the container and suitable for the injection, preservation and thawing of a small volume bio-specimen. <P>SOLUTION: This container for the freeze preservation is equipped with concretely a container main body consisting of a flexible resin, an opening part communicating with the inside of the container main body and sealable by welding, and a seal-opening part communicating with the inside of the container main body by opening the seal. The method of freeze-preserving by using the above container is also provided. By using the container for freeze preservation, it is possible to perform the preservation of rare bio-specimens safely and easily. Further in detail, since the container for freeze preservation is hardy invaded by the liquid nitrogen, sundry bacteria or viruses to the inside of the container, the bio-specimen preserved at the inside of the container can not be contaminated. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a cryopreservation container for biological samples. Furthermore, the present invention relates to a cryopreservation method using the cryopreservation 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 (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 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 vial formed of a film of an ethylene-vinyl acetate copolymer that has been irradiated with an electron beam and biaxially stretched. Patent Document 5 discloses a cryopreservation vial formed of a biaxially stretched crosslinked polyethylene film.

  In order to cryopreserve floating cells using such a bag, first, a solution in which the cells are suspended is prepared, and 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. A cryovial is a typical vial in which a lid is screwed into a vial opening as shown in FIG. The cryovial is used by storing a biological sample in a container, screwing and attaching a lid to the opening, and immersing it in liquid nitrogen for storage.

  However, when a cryovial containing a biological sample is stored in liquid nitrogen, liquid nitrogen or various germs (mainly mycoplasma) present in the liquid nitrogen enter the inside of the container from the gap between the container body and the lid of the cryovial. There is a fear. The gap is caused by the heat shrinkage of the resin during the molding of the container body and the lid. If molding is performed so that such a gap does not occur, not only the manufacturing cost becomes very expensive, but also the resin may be scraped off due to friction between the lid and the container body.

  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 may burst or the lid may be fired, resulting in an accident in which the operator is injured. If an operator is injured in such an accident, liquid nitrogen bacteria may enter from the operator's wound and develop into a biohazard.

  Furthermore, not only the germs in the liquid nitrogen, but also when a human-derived biological sample contains hepatitis, AIDS virus, etc., it infects other biological samples from a container in which cells with viruses are frozen during freezing. There is also a fear.

  As means for solving such problems, it is conceivable to use the bags of Patent Documents 1 to 5. However, since the bags of Patent Documents 1 to 5 have a large volume, they are not necessarily suitable for storing a small volume of biological sample. This is because the bag needs to have a port for introducing the dispersion liquid of the biological sample, and the inner volume of the bag needs to be increased accordingly. Then, it is necessary to dilute the biological sample with a storage solution or the like in order to make the capacity enough to be stored in the bag. As a result, when using the sample after cryopreservation, it is necessary to perform a complicated operation of concentration.

  Another solution is to use a commercially available thermoplastic tube. Specifically, after the cryovial is inserted into the thermoplastic tube, it is shrunk by heat to prevent liquid nitrogen from being mixed. However, since the heat-shrinkable tube is heated to near 100 ° C. with a heat gun or the like, the cells in the container may be killed by heat. The tube opening is pinched and sealed with tweezers heated with a heat gun, and the tube is often damaged at this time. For this reason, these products are not very popular.

  Furthermore, as another solution, a method of storing in an air layer of liquid nitrogen or an ultra-low temperature freezer can be considered. However, when cells are stored for a long period of time, each time the freezer or tank lid is opened and closed in the air layer, a temperature change occurs, which causes a problem in reliability.

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 injecting, storing, and taking out a small-volume biological sample without liquid nitrogen, bacteria, or viruses entering the container.

The present invention
[1] A container for cryopreserving a biological sample,
A container body;
An opening that communicates with the interior of the container body and can be sealed by welding;
A cryopreservation container having an opening portion communicating with the inside of the container body by opening the container,
[2] The cryopreservation container according to [1], wherein the opening can be connected to an injection tool and has a structure capable of exhausting air inside the container body.
[3] The cryopreservation container according to [1], wherein the opening portion has a constricted portion structure that is broken by torsion,
[4] A method for cryopreserving a biological sample using a cryopreservation container,
The cryopreservation container includes a container body,
An opening that communicates with the interior of the container body and can be sealed by welding;
Comprising an opening that communicates with the interior of the container body by opening the container;
Introducing a biological sample from the opening, and storing the biological sample in a container body;
Sealing the opening by welding;
A cryopreservation method comprising a step of storing the biological sample contained in the container at a cryogenic temperature, and
[5] Furthermore, a step of thawing the stored biological sample;
Removing the biological sample by opening the opening;
And a step of taking out the stored biological sample.

  According to the cryopreservation container of the present invention, a rare biological sample can be stored safely and easily. More specifically, in a cryopreservation container, liquid nitrogen, germs, or viruses do not easily enter the container, so that biological samples stored in the container are not contaminated. And since self by liquid nitrogen can be prevented, there is no possibility that an operator may be injured.

  The present invention will be described below with reference to the drawings.

  FIG. 1 shows a cryopreservation container according to the present invention. “Cryopreservation container” refers to a container that can contain a biological sample, and is a container that is made of a material that can withstand physical strength in an environment of at least 0 degrees or less. Furthermore, it refers to a container in which liquid nitrogen, germs or viruses do not easily enter the container during cryopreservation. The structure of the cryopreservation container according to the present invention can be communicated with the container body 1 made of a flexible resin, the inside of the container body, the sealable opening 2, and the inside of the container body by opening the container. The opening part 3 is provided.

The “biological sample” is not particularly limited as long as it is derived from a living body, and includes animal tissues and microorganisms. For example,
i) blood components such as whole blood, red blood cells, white blood cells, plasma, platelets and platelet-rich plasma; ii) rare cells such as bone skeleton mononuclear cells, hematopoietic stem cells, ES cells, mesenchymal stem cells, sperm cells and egg cells;
iii) Microorganisms including microbial mutants such as drug-resistant Mycobacterium tuberculosis iv) Sperm and eggs. Among these biological samples, ii) rare cells and iv) sperm and ovum, particularly preferably iv) sperm and ovum, from the viewpoint of storing a small amount.

  “Frozen storage” means 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. (under cryogenic temperature), more preferably −80 ° C. or less. Preferably, it means storing in an environment of -196 degrees (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 main body” refers to a container having a general-purpose container shape having a space (housing) for accommodating a biological sample.

  The container body 1 is provided with an opening 2. The “opening” is a structure that communicates with the inside of the container body 1, and a biological sample can be introduced into the container body 1, and the inside of the container body 1 is sealed by a sealing operation by welding. The part that can be done. The top view has, for example, a flat pupil shape as shown in FIG. That is, the opening 2 of the present invention serves as a sealing lid in the cryopreservation container. Since the opening 2 is sealed by welding, liquid nitrogen or the like does not enter the container body.

  The opening 2 can be connected to an injection tool such as a syringe or pipette so that the biological sample can be easily introduced into the container body. A structure for exhausting is preferable. For example, as shown in FIG. 3, a structure (injection tool connection structure 21) having a substantially circular shape to which the tip of a syringe can be connected is provided. Can be exhausted.

  The container body 1 containing the biological sample seals the opening 2 after extracting the injection tool. The method for sealing will be described later.

  Further, the container body 1 is provided with an opening portion 3. The “opening portion” in the present invention refers to a portion that can be removed by breaking, shearing, fusing and cutting, preferably by human force. Furthermore, the part which can open | release the inside of the container main body 1 by removing and can take out the biological sample accommodated in the inside of the container main body 1 is said. The opening part 3 plays the role of a lid for opening for taking out the biological sample accommodated in the container body. For example, the constricted part 31 (FIG. 4) fractured | ruptured by torsion, the notch part 33 (FIG. 5) fractured | ruptured by a notch, etc. are mentioned. In particular, it is preferable to provide a handle portion 32 as shown in FIG.

  Further, the position where the opening part 3 is provided is not particularly limited as long as it does not hinder the introduction of the biological sample from the opening part 2 into the container body 1, but the position where the opening part 2 is paired with the container body 1. It is preferable to provide in.

  The container main body 1, the opening part 2, and the opening part 3 are integrally comprised with flexible resin. In other words, the material constituting the cryopreservation container of the present invention is made of a flexible resin. The “flexible resin” in the present invention refers to a resin that is flexible enough to at least seal the opening by thermal welding. Examples of such a resin include polypropylene, polyethylene terephthalate, polyethylene, and ethylene-vinyl acetate copolymer.

further,
・ Excellent physical strength at cryogenic temperatures ・ Low thermal insulation that does not impede heat conduction into the container body during cryopreservation ・ Material that satisfies at least one of the requirements of being able to break due to stress concentration due to torsion Preferably there is. Examples of materials that satisfy these requirements include, but are not limited to, polyethylene and ethylene-vinyl acetate copolymers.

  The shape of the cryopreservation container of the present invention is not particularly limited and can be appropriately set by those skilled in the art depending on the ease of cryopreservation and the nature of the flexible resin. For example, examples of the shape of the container body 1 include a columnar shape, a prismatic shape, a conical shape, and a pyramid shape. Furthermore, in order to improve the handling properties, for example, a concavo-convex structure that exhibits an anti-slip effect may be provided around the container body 1. 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. The outer diameter of the container body 1 at this time is about 1 to 50 mm, preferably about 10 to 15 mm, but the present invention is not limited to this.

  Further, the internal volume of the container body 1 is not particularly limited, but the shape is similar to that of a cryovial that is used to those skilled in the art, and a viewpoint that can be applied to accessories such as a cane for attaching the cryovial. To about 0.5 to 5.0 ml, preferably about 1.0 to 2.0 ml, but is not limited thereto. Further, the thickness of the container body 1 is not particularly limited as long as it does not hinder heat conduction in cryopreservation, and is, for example, about 0.3 to 2 mm, preferably about 0.4 to 1 mm. .

  In addition, the shape inside the container body faces the opening part 3 so that when opened by breaking the opening part 3, the biological sample contained in the container body 1 is not scattered and no residual liquid remains. It is preferable to provide a tapered portion 11 that converges in a tapered shape. The width of the converged end portion is about 0.1 to 5 mm, preferably about 1 to 3 mm, considering that the biological sample accommodated inside the container body can be safely taken out without scattering. The invention is not limited to this.

  The method for producing a cryopreservation container according to the present invention can be implemented very easily because the container body 1, the opening 2 and the opening 3 are integrally formed of a flexible resin. For example, injection molding, extrusion molding, blow molding and the like can be mentioned. Among these manufacturing methods, blow molding is preferable from the viewpoint of easy production and low cost, but the present invention is limited to this. is not.

The present invention further extends to a method for cryopreserving biological samples using the cryopreservation container of the present invention. In particular,
1) A biological sample is introduced from the opening 2 and the biological sample is accommodated in the container body 1 Step 2) The opening 2 is sealed by welding 3) A biological sample accommodated in the container body 1 Storing at a cryogenic temperature.

  First, 1) a biological sample is introduced from the opening 2 and the biological sample is accommodated in the container body 1. The method for accommodating the biological sample in the container body 1 can be appropriately selected by those skilled in the art depending on the type of biological sample to be stored. For example, when storing cells such as eggs, sperm and ES cells, or microorganisms such as drug-resistant M. tuberculosis, prepare the cell suspension by suspending the cells in a medium or cell storage solution that enables cryopreservation. After that, the container body 1 can be accommodated. Here, the accommodation can be performed using an injection tool such as a syringe and a pipette. In addition, it is preferable that the structure of the opening 2 as shown in FIG. 3 can be connected to the syringe and that the air inside the container main body is exhausted because the work becomes easy.

Here, the cell concentration in the cell suspension solution can be appropriately determined by those skilled in the art depending on the type of cell, and is not particularly limited. For example, when the cell is a sperm, the concentration is about 2 × 10 ⁷ to 2. × 10 ⁸ cells / ml, and 1 to 10 cells / ml for an egg. 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.

  Next, 2) the opening 2 is sealed by heat welding. 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 flexible resin. For example, when the flexible resin is polyethylene terephthalate, it is about 150 to 300 ° C., preferably about 190 to 250 ° C. from the viewpoint of welding strength. For example, when the flexible resin is polyethylene, the temperature is about 100 to 250 ° C., preferably about 150 to 200 ° C. from the viewpoint of welding strength. Thus, the opening 2 sealed by heat welding no longer allows liquid nitrogen, germs or viruses to enter the container body.

  3) The biological sample housed in the container body 1 is stored frozen. As described above, the temperature condition at the time of cryopreservation refers to storage in an environment of 0 ° C. or less, but from the viewpoint of storage over a long period of time, it is preferably −20 ° C. or less (very low temperature), more preferably − It is in an environment of 80 degrees or less, particularly preferably -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.

Furthermore, the cryopreservation method of the present invention comprises:
4) Thawing the stored biological sample 5) Opening the unsealed portion 3 to extract the internal biological sample 6) Extracting the stored biological sample.

  When using a cryopreserved biological sample, first 4) thaw the stored biological sample. The method of thawing can be appropriately selected by those skilled in the art and is not particularly limited. For example, the thawing can be performed by a warm bath at about 37 ° C. At this time, since the opening is sufficiently sealed, there is no liquid nitrogen in the container body, so there is no damage to the container due to rapid expansion of liquid nitrogen, and the operator may be injured accordingly. Absent. Furthermore, there is no risk of biohazards due to biological samples.

  Next, the biological sample is thawed in a container with the cells taken out in a 37 ° C. warm bath. At this time, unlike the cryovial, the product of the present invention is completely sealed, so that the water in the warm bath can be thawed without entering the container. After thawing the biological sample, the surroundings of the container are wiped with a disinfectant solution, and 5) the cells can be taken out by opening the opening 3. The opening can be appropriately selected by those skilled in the art depending on the structure of the opening 3. For example, when the opening portion 3 has a structure 31 that can be broken by threading, the opening is achieved by breaking by threading. Moreover, when the opening part 3 is a structure provided with the notch part 33, opening is achieved by the fracture | rupture by a notch. As described above, it is preferable to easily open by breaking by human force, but it may be opened by auxiliary shearing, fusing by heat or laser.

  Finally 6) Remove the thawed biological sample. The removal operation can be appropriately selected by those skilled in the art and is not particularly limited. For example, since the container main body has flexibility, the biological sample accommodated like a dropper is pressed by pressing the container main body. It can be taken out. Further, for example, the air hole may be provided by shearing the vicinity of the other end of the container main body 2 with respect to the place where the opening portion 3 is provided with a scissors or the like.

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

  FIG. 1 shows a cryopreservation container of the present invention. The cryopreservation container of FIG. 1 includes a container main body 1, an opening 2 that communicates with the inside of the container main body 1 and can be sealed by heat welding, and the inside of the container main body 1 by opening the container. The opening part 3 which connects is provided. The container body 1, the opening 2 and the opening 3 are all manufactured by blow molding of an ethylene-vinyl acetate copolymer. Here, the internal shape of the container body 1 is provided with a tapered portion 11 that converges in a tapered shape toward the opening portion 3. Further, the sectional view of the opening 2 has a pupil shape (FIG. 2). Furthermore, the opening part 3 is the constriction part 31, as shown in FIG. 5, and the operator can break and remove the opening part 3 (the constriction part 31 and the handle part 32) by twisting the handle part 32. .

  FIG. 3 is a modification of the upper surface of the opening 2. The opening 2 in FIG. 3 is provided with an injection tool connection structure 21 and a communication structure 22. The broken line shows the injection tool connection structure 21.

  FIG. 5 is a modification of the opening part 3. The unsealing part 3 of FIG. 5 has a notch 33, and can be broken and removed by the notch.

  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, liquid nitrogen, various bacteria or viruses are unlikely to enter the inside of the container body 1, so that the stored biological sample 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. 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 the cryopreservation container of this invention. It is an upper surface enlarged view of the opening part 2 of FIG. FIG. 6 is an enlarged top view showing a modification of the opening 2. The broken line shows the injection tool connection structure 21. It is an enlarged view of the opening part 3 of FIG. The broken line shows the tapered portion 11 inside the container body. It is a figure which shows the modification of the opening part. The broken line shows the tapered portion 11 inside the container body. It is a figure which shows the conventional cryopreservation container.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container body 11 Taper part 2 Opening part 21 Injection tool connection structure 22 Communication structure 3 Opening part 31 Constriction part 32 Handle part 33 Notch part 4 Screwing structure

Claims (5)

A container for cryopreserving a biological sample,
A container body;
An opening that communicates with the interior of the container body and can be sealed by welding;
A cryopreservation container comprising an opening portion that communicates with the inside of the container body by opening the container.   The cryopreservation container according to claim 1, wherein the opening is connectable to an injection tool and has a structure capable of exhausting air inside the container body.   The cryopreservation container according to claim 1, wherein the opening portion has a constricted portion structure that is broken by twisting. A method for cryopreserving a biological sample using a cryopreservation container, comprising:
The cryopreservation container includes a container body,
An opening that communicates with the interior of the container body and can be sealed by welding;
Comprising an opening that communicates with the interior of the container body by opening the container;
Introducing a biological sample from the opening, and storing the biological sample in a container body;
Sealing the opening by welding;
A cryopreservation method comprising: storing a biological sample contained in the container at a cryogenic temperature. A step of thawing the stored biological sample;
Removing the biological sample by opening the opening;
And taking out the stored biological sample. The cryopreservation method according to claim 4.

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