JP3495075B2 - Cryopreservation container - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a container for cryopreservation used in the medical, cultivation, and food fields, and particularly to animals and plants or human tissues or cells as they are. -196 ° C
The present invention relates to a container for cryopreservation that can be stored for a long time at extremely low temperatures. [0002] Biomaterials such as skin pieces, bones, heart valves, kidneys, nerves and blood vessels are used at -80 ° C in the food field.
At a low temperature of −90 ° C., the tissue cannot be stored for a long period of several months without destroying the tissue. In recent years, glycerin or DMSO (Dimethyl sulfo) has been used.
(xide) or the like, and immersion in a culture solution to which a cryoprotective solution has been added, and cryopreservation at about -196 ° C. [0003] As a cryopreservation container, there is disclosed a cryogenic temperature-resistant container in which the inner layer is made of ultra-high molecular weight non-stretched polyethylene and the outer layer is heat-sealed between the inner layers of a laminated material made of biaxially stretched high-density polyethylene (see, for example, JP-A-2003-133873). Tokiko Sho 60-4
9429). Further, there is disclosed an ultra-cryogenic container in which an inner layer is formed of a two-layer sheet in which an extremely high molecular weight unstretched polyethylene is formed and an outer layer is formed of a biaxially stretched high-density polyethylene terephthalate (JP-A-2-136).
No. 140). [0004] However, the above-mentioned container is a container for a liquid material, and only a cylindrical opening is described therein, and a living tissue such as a skin piece, a bone, a heart valve, a kidney, a nerve, a blood vessel, or the like is directly used. There was a problem that it was not possible to insert in the shape of. Further, as a container for cryopreservation, a synthetic resin bag provided with an occlusal tool having an occlusal portion at an opening has been disclosed (Japanese Patent Publication No. 49-18964). However, there has been a problem that the air adhering to the tissue surface cannot be completely removed after the opening is heat-sealed, and further, it cannot be used for cryopreservation. [0006] Furthermore, the fresh food is melted at a temperature of -3 ° C.
A cryopreservation method of a fresh food product, which is sealed in a bag together with an aqueous solution at 10 ° C. and then frozen, is disclosed (Japanese Patent Publication No. 1-15272). However, in this method, a bag whose entire circumference is heat-sealed is used, and when applied to a living tissue, there is a problem that air adhering to the tissue surface appearing after the heat sealing cannot be removed. If there is such a residual air, the expansion of the air can <br/> and was rapidly thawed, liquid leakage occurs in heat-sealed portion, there is a problem that sterility of the biological tissue can not be maintained. SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and has as its object to easily insert an object to be preserved, such as a living tissue, into a bag, and to be bulky during cryopreservation. The residual air in the container can be easily and completely removed in a state where the object to be preserved is sealed, and furthermore, -19
An object of the present invention is to provide a cryopreservation container that does not peel or break the heat-sealed portion even when thawed after storage at an extremely low temperature of 6 ° C. and does not cause leakage of the culture solution. [0008] A cryopreservation container according to the present invention is a bag-like container formed of a laminate having a specific resin film as an inner layer and an outer layer, and an opening portion is provided with an occlusal portion. Is provided, and a container for cryopreservation in which a gas permeable filter is provided in at least one portion of a peripheral portion, wherein the object to be stored is
When the air remaining in the container is sealed,
After being discharged from the heater, the above-mentioned opening, occlusion and breathability
The part that contains the filter and the other part
And then heat-sealed
Cut into two parts, the opening, bite and
After the breathable filter has been disconnected from the container,
Characteristic is that the container containing the stuff is stored at cryogenic temperature
And As the inner layer, a resin film or sheet of unstretched polyethylene is used. It is not preferable to use stretched polyethylene as the inner layer, because stretching is returned by heat sealing or shrinkage occurs in the heat sealed portion. When the thickness of the resin film or sheet used for the inner layer is small, a predetermined strength cannot be obtained, and when the thickness is large, rapid freezing becomes difficult.
mm is preferred. As the outer layer, biaxially oriented polyethylene terephthalate, uniaxially oriented polyethylene naphthalate,
Undrawn polyethylene naphthalate, polyparabanic acid, perfluoroalkoxy resin, polytetrafluoroethylene,
A resin film or sheet of an ethylene-tetrafluoroethylene alternating copolymer, a tetrafluoroethylene-hexafluoropropylene copolymer, an ethylene chloride trifluoride resin, an aromatic polyimide or a biaxially oriented high-density polyethylene is used. [0012] The thickness of the resin film or sheet used for the outer layer is set to 0.01 for the same reason as for the inner layer.
2 to 0.5 mm is preferred. [0013] The inner layer and the outer layer are laminated by dry lamination with an adhesive such as polyester, urethane or epoxy to obtain a laminate. Incidentally, in order to improve the adhesive strength, a corona discharge treatment, a plasma treatment, a chemical treatment, or the like may be performed on the adhesion surfaces of the resin films or sheets used for the inner layer and the outer layer. In the case where a bag-like container is formed by using the above-mentioned laminate, the laminates are overlapped so that the inner layers thereof are in contact with each other, and then the peripheral portions of three sides where the cut portions of the laminate overlap each other are formed. One side is an opening, and the other two sides are heat-sealed. The shape of the cryopreservation container of the present invention may be a bag shape having a flat bottom such as a gusset type or a ship bottom type, in addition to a bag shape obtained by stacking two of the above laminates. . The air-permeable filter is provided for discharging the residual air in the cryopreservation container to the outside of the container. Examples of the filter include polyolefin and polyfluoride having fine pores permeable only by air. A resin film of vinylidene or tetrafluoroethylene is preferable, and its pore size is preferably 0.22 μm or less so as not to transmit bacteria and a cryopreservation solution. The mounting position of the breathable filter is designed to provide at least one location of the periphery of the vessel, actually slightly downward preferred occlusal portion described later. How to mount the air-permeable filter, for example, that a hole penetrating the inner and outer layers of the cryopreservation container and location mounting breathability filter so as to close the hole from the outer layer of the container is heat sealed Is mentioned. When the cryopreservation container is suspended and stored, a hole for suspending the container may be provided in the peripheral portion. [0019] Use for how the cryopreservation container, as shown in FIG. 1, first, put preservation object 11 an opening 5 make a cryopreservation container 6 in the above, followed by DM
The cryopreservation liquid such as SO is filled up to slightly below the bite portion 4. Next, the occlusal portion 4 is closed, and the remaining air in the container 6 is discharged from the air permeable filter 9 provided below the occlusal portion 4 to adjust the capacity of the container 6. This capacity adjustment is performed in order to prevent the thickness of the container 6 from becoming unnecessarily large, so that rapid freezing cannot be performed. After the capacity of the container 6 has been adjusted, as shown in FIG. 2, heat sealing is performed so that no air remains in the container below the heat sealing portion 10.
The opening 5, the occlusal part 4, and the gas permeable filter 9 are cut off from the container 6 by cutting the zero into two. Next, the container 6 containing the object to be preserved 11 is stored in a freezer or the like. As a freezing method, it is preferable to put the container 6 in a program freezer and freeze while controlling the temperature. When the object to be preserved 11 is a thin material such as skin, the object to be preserved is not directly put in the container 6 but is attached to a plastic plate or the like which can withstand freezing similarly to the container 6.
It is preferable to store the whole plate. The thickness of the plastic plate used at this time is 100-2,000 μm
Is preferred. An embodiment of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic diagram showing the entire configuration of the cryopreservation container, and FIG. 2 is a schematic diagram showing a state in which the object to be preserved is sealed in the cryopreservation container. First, as shown in FIG. 1, a laminate of an inner layer 1 made of a non-stretched ultra-high-density polyethylene film and an outer layer 2 made of a biaxially stretched polyethylene terephthalate film is placed so that the inner layers 1 are in contact with each other. After overlapping
Using a heat fusion machine (Fuji Impulse "Policra"), heat-sealing a polyethylene ribbon-like chuck member (Idemitsu Petroleum Co., Ltd. "Polyloc") to both inner layers 1 on the overlapping side 3a. An opening 5 having an occlusal portion 4 was formed. The opening 5 is preferably provided over the entire length of the side 3a in order to facilitate taking in and out of the object to be preserved. Next, the inner layer 1 was heat-sealed on each of the two sides 3b and 3c where the cut portions of the laminate were overlapped with each other by a heat fusion machine to form a bag-like container 6. Next, the non-wetted part 7 was provided by heat-sealing a slightly inner side of the folded side 3d. In order to obtain a predetermined heat seal strength, the heat seal width is preferably about 5 mm. The bag-shaped container 6 can be manufactured to any size by appropriately selecting the size of the laminate.
The shape of the bag-shaped container 6 is, for example, from the convenience in handling, for example,
Those having autonomy such as a tubular shape are preferred. Next, an oval-shaped hole (not shown) is opened slightly below the occlusion portion 4, and a gas-permeable filter 9 laminated with Teflon and polyethylene is heat-sealed so as to close the hole, and a non-liquid contact portion is formed. 7 was also provided with a circular hole 8 to obtain a cryopreservation container 6. Evaluation of the performance of the cryopreservation container The object to be preserved (skin) 11 cut from the animal's corpse was immediately stapled on a plastic plate (not shown) with a stapler so that the end would not curl. After the plate was inserted into the cryopreservation container (not shown), the bite portion 4 was closed. The above-mentioned container for cryopreservation is replaced with a gas permeable filter 9.
Was placed on the upper side, and an appropriate amount of vibration was applied to move all the air adhering to the skin to the upper part of the container 6 and separated. Next, the occlusal part 4 of the container 6, the air-permeable filter 9,
The upper part of the container 6 containing the whole amount of air and a part of the culture solution is heat-sealed with a constant width so as to bisect the lower part of the container 6 containing the object to be preserved 11, and the heat seal part 10 is cut and divided into two parts. 6 is separated from the upper part and the lower part,
A cryopreservation container without any residual air on one side was obtained. Thereafter, the cryopreservation container enclosing the object to be preserved (body skin) 11 is kept in a refrigerator at 4 ° C. for 2 hours,
After freezing to −80 ° C. with a program freezer, a metal hook was hooked on the hole 8 and immediately immersed in liquid nitrogen for cryopreservation. After storing in liquid nitrogen for 24 hours, the cryopreservation container was taken out and immediately thawed in warm water at 37 ° C. No peeling or breakage of the heat-sealed portion was observed, and no leakage of the culture solution was observed. The structure of the cryopreservation container of the present invention is as described above, and the air remaining in the container is easily and completely removed from the air-permeable filter in a state in which the object to be preserved is sealed. After storage at a very low temperature of -196 ° C, even if thawed, peeling and breakage of the heat-sealed portion and leakage of the culture solution do not occur, and the preserved material can be stored under aseptic conditions. Alternatively, it is suitable for long-term storage of human tissues or cells. In addition, since there is no spillage of the culture solution from the enclosing of the preserved material to the freezing, it is sanitary.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view showing a cryopreservation container of the present invention. FIG. 2 is a perspective view showing a state in which an object to be preserved is sealed in the container for cryopreservation of the present invention. [Description of Signs] 1 Inner layer 2 Outer layers 3b, 3c, 3d Overlapping side 4 Occlusion portion 5 Opening 6 Bag-shaped container 7 Non-wetted portion 8 Hole 9 Gas permeable filter 10 Heat seal portion 11 Stored object

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI B65D 33/25 B65D 85/50 A 85/50 A61J 1/00 331C (58) Investigated field (Int.Cl. ⁷ , DB name) ) A61J 1/00-3/10 B65D 33/25 B65D 85/50

Claims (1)

(57) [Claims] [Claim 1] Biaxially stretched polyethylene terephthalate, uniaxially stretched polyethylene naphthalate, unstretched polyethylene naphthalate, polyparabanic acid, perfluoroalkoxy resin, polytetrafluoroethylene Outer layer of any one selected from ethylene, ethylene-tetrafluoroethylene alternate copolymer, tetrafluoroethylene-hexafluoropropylene copolymer, ethylene chloride trifluoride resin, aromatic polyimide and biaxially oriented high-density polyethylene a bag-like container formed from a laminate and, in the opening occlusal portion is provided, and at least one location breathable filter is not that frozen storage vessel provided in the peripheral portion, the With the preserved material enclosed in the container
After the residual air has been exhausted from the breathable filter,
The part including the opening, the bite, and the breathable filter
Heat seal so that the other parts are bisected.
And then cut so that the heat seal is bisected.
The opening, the bite and the breathable filter
After being separated from the container, the container containing the
A cryopreservation container characterized by being stored at a very low temperature .