WO2012099249A1

WO2012099249A1 - Low temperature storage method and low temperature storage container

Info

Publication number: WO2012099249A1
Application number: PCT/JP2012/051244
Authority: WO
Inventors: 木野　正人; 昭夫清水
Original assignee: 株式会社ミツヤコーポレーション; 学校法人創価大学
Priority date: 2011-01-20
Filing date: 2012-01-20
Publication date: 2012-07-26
Also published as: JP5958913B2; CN103327839B; JPWO2012099249A1; CN103327839A

Abstract

A low temperature storage container (10) is sealed, after a stretchable middle bag (12) that makes a subject to be stored (A) exist therein and is filled with a liquid (C) having the compression rate larger than a liquid (D) and having the freezing point temperature lower than the liquid (D) is put into an outer container (11), and a space between the outer container (11) and the middle bag (12) is filled with the liquid (D) with the volume which is increased when freezing. Then, the low temperature storage container (10) is cooled, and the subject to be stored (A) is stored at the storage temperature that is the temperature between the freezing points of the liquid (C) and the liquid (D) and is lower than the freezing point temperature at atmospheric pressure of a liquid in the subject to be stored (A).

Description

Cryogenic storage method and cryogenic storage container

The present invention relates to a cryopreservation method for preserving preservation objects such as foods, organs and cells at low temperatures, and a cryopreservation container used in this cryopreservation method.

Generally, organic substances such as foods, organs and blood can suppress the activity and chemical reaction of bacteria and enzymes, and the storage time becomes longer as the storage temperature is lower. Therefore, when storing an object to be stored for a long time, it is often stored frozen by freezing, amorphous solidification, or the like.

However, in cryopreservation, the tissue is damaged by volume expansion due to the formation of ice crystals. Therefore, there is a need for a method for storing a storage object at as low a temperature as possible while suppressing the formation of ice crystals.

In addition, a method of adding an antifreeze agent to concentrate and solidify an organ or tissue has been studied, but it can be applied only to a thin organ or tissue, and it is necessary to remove the added antifreeze agent. Therefore, there is a demand for a method for storing organs and cells without adding a drug such as an antifreezing agent.

By the way, the freezing point of water decreases by about 1 ° C. every time the pressure increases by 10 MPa up to about 200 MPa. That is, in order to decrease the storage temperature in the non-freezing state by 1 ° C., the pressure may be increased by about 10 MPa.

Therefore, in order to suppress damage due to ice crystals, a pressure freezing is performed in which a high pressure of about 200 MPa is applied to the pressure vessel using a pressure device, and the object to be stored is kept in a non-freezing state at about −20 ° C. A law has been proposed. In addition, when preparing a microscope sample, the object to be stored may be frozen by reducing the pressure to normal pressure. Furthermore, a high pressure of 400 to 500 MPa may be applied using a pressurizing device to denature proteins and enzymes such as foods and may be stored in a non-frozen state at a low temperature.

However, the conventional pressure freezing method requires a large-scale pressure device that generates high pressure.

Therefore, there has been proposed a freeze pressurization method in which water in the pressure vessel is frozen and the pressure in the pressure vessel is increased by utilizing volume expansion when water is frozen. In this freeze pressurization method, no special pressurizing device is required, and a high pressure can be easily generated.

For example, in Patent Document 1, food and storage water are put in a sealed container (pressure container) to freeze part of the stored water, and the pressure in the sealed container is increased from atmospheric pressure by volume expansion of the generated ice. Thus, a technique for storing food at a low temperature in a non-freezing state at a temperature below the freezing point at normal pressure is disclosed.

Further, Non-Patent Document 1 discloses that in a non-freezing state, water or an aqueous solution is filled in a pressure-resistant container containing food, frozen at a sub-freezing temperature, and high pressure is applied in the pressure-resistant container. A technique for impregnating food with seasoning water is disclosed.

Japanese Patent Laid-Open No. 06-303954

However, the conventional freeze pressurization method requires an expensive pressure vessel. And, if the amount of ice exceeding the expected amount is generated, such as when the temperature in the pressure vessel is too low, a high pressure is suddenly applied in the pressure vessel, so the pressure vessel may be damaged, An expensive pressure vessel needs to be repaired or remanufactured. Moreover, the pressure vessel is heavy and difficult to carry.

Therefore, there is a demand for a container that is light in weight, easy to handle and hard to break so that it can be easily transported.

In addition, when the amount of freezing is larger than expected, or when the strength is insufficient due to the material, thickness and shape of the pressure vessel, the pressure vessel may be damaged.

In view of the above points, the present invention provides a low-temperature storage method in which an excessively high pressure is not applied, and a low-temperature storage container suitable for this low-temperature storage method, while relieving abrupt changes in the internal pressure of the container containing the storage object. The purpose is to provide.

The cryopreservation method of the present invention has a stretchable first closed space filled with a first liquid that increases in volume when solidified, a compressibility greater than that of the first liquid, and is higher than that of the first liquid. Forming a retractable second closed space filled with a second liquid having a low-temperature freezing point in the container, placing a storage object in the container, and first and second closures A step of sealing the container in which the space is formed and the object to be stored is sealed; and after sealing the container, the first liquid and the second liquid are cooled to a temperature lower than the freezing point of the first liquid. Storing the storage object at a storage temperature lower than the freezing point of the liquid in the storage object at the atmospheric pressure, the temperature being between the freezing point of the liquid and the liquid.

According to the low-temperature storage method of the present invention, after the container is sealed, when the first liquid is cooled to a temperature lower than the freezing point of the first liquid, at least a part of the first liquid is solidified (frozen). Since the first closed space occupied by the first liquid expands due to the increase in volume when the first liquid solidifies, the volume in the container other than the first closed space decreases, so that the pressure in the container is reduced. Ascend and exceed atmospheric pressure.

At this time, since the second liquid filled in the second closed space has a higher compressibility than the first liquid, a part of the expanded volume of the first closed space is equal to the volume of the second closed space. Absorbed by reduction. And since the freezing point of the second liquid is lower than the storage temperature, it does not freeze even when cooled to the storage temperature. Therefore, compared with the case where the second closed space filled with the second liquid is not formed in the container, the rapid increase in pressure in the container is suppressed and the pressure increase becomes slow.

Therefore, there is no risk of the container being unintentionally damaged, and it is not always necessary to use a pressure vessel as the container. And even when using a pressure vessel as a container, since a wall thickness may be thin and pressure resistance performance may be low, a lightweight and cheap pressure vessel can be used.

Furthermore, according to the low temperature storage method of the present invention, the upper limit value of the pressure in the container and the pressure increase mode according to the upper limit of the internal volume of the container, the amount of the first liquid, the amount of the second liquid, and the cooling mode. Is uniquely determined. Therefore, there is no possibility that the container will be damaged due to an increase in pressure more than expected, and the object to be stored can be stably stored at a low temperature in a non-frozen state.

In the conventional freeze pressurization method, for example, as disclosed in Non-Patent Document 1 described above, a seasoning liquid is filled in a pressure-resistant container containing a food surrounded by a seasoning liquid, and the aqueous solution is frozen at −25 ° C. A high pressure of several hundred MPa is applied. However, in this method, the food is frozen only by a slight change in the ratio between the aqueous solution concentration and the seasoning solution concentration.

In the low-temperature storage method of the present invention, unlike the conventional concentration adjustment, a desired high pressure is obtained by adjusting the amount of liquid, and a desired high pressure can be stably obtained as compared with the conventional method.

Furthermore, in the low-temperature storage method of the present invention, the freezing point of the first liquid is lowered by pressurization, and a drug such as a freezing point lowering agent for lowering the freezing point is not required. However, a chemical such as a freezing point reducing agent may be added to the first liquid.

In the cryopreservation method of the present invention, for example, the object to be preserved may be contained in a space other than the first and second closed spaces in the container, and the space may be filled with a liquid having a freezing point lower than the preservation temperature. .

Further, for example, the second closed space is included in the first closed space, the object to be stored is included in the second closed space, and the temperature other than the first closed space in the container is lower than the storage temperature. A liquid having a freezing point of may be filled.

Furthermore, for example, the internal space in the container is the first closed space, the second closed space may be included in the first closed space, and the storage object may be included in the second closed space. .

In the low-temperature storage method of the present invention, in the step of forming the first and second closed spaces in the container, the second closed space is formed in the first closed space, and the storage object is stored in the container. In the internalizing step, the object to be stored is in the second enclosed space, and in the step of increasing the pressure in the container, at least a part of the first liquid is frozen to produce an icing capsule, It is preferable to increase the pressure in the closed space of 2 to a pressure exceeding the atmospheric pressure.

In this case, the object to be stored is contained in the first closed space filled with the first liquid. Therefore, an object to be stored is contained in an icing capsule formed by freezing the first liquid. Therefore, the storage object can be stored at a low temperature in the frozen capsule.

Conventionally, there has been a method for cryopreserving the object to be stored in the freeze capsule, but it has not been possible to stably preserve the object in the freeze capsule in a non-frozen state. The reason is considered to be that when the ice thickness is within a practical range, cracks occur in the frozen capsule due to an increase in internal pressure, the pressure in the frozen capsule decreases, and the object to be stored freezes.

On the other hand, according to the low-temperature storage method of the present invention, the second liquid serves as a buffer material, and a rapid pressure increase in the frozen capsule is suppressed. Therefore, since cracks do not occur in the frozen capsule, the pressure in the frozen capsule does not decrease, and the storage object can be stably stored at a low temperature in a non-frozen state.

Therefore, in the low-temperature storage method of the present invention, in the step of forming the first and second closed spaces in the container so that the frozen capsule is maintained in the step of storing the storage object, the first liquid and the first liquid It is preferable to set the volume ratio with respect to the second liquid.

Moreover, in the low-temperature storage method of the present invention, it is preferable that the method further includes a step of forming a third closed space filled with a liquid or a gas to be contained in the second closed space.

In this case, the storage object contacts only the liquid or gas filled in the third closed space and does not contact the second liquid. Therefore, even if the storage object is a food, organ, cell, or the like, the liquid that denatures the storage object can be filled in the second space, and the options for the second liquid are widened.

Further, in the low temperature storage method of the present invention, it is preferable that the first closed space is filled with water as the first liquid in the step of forming the first and second closed spaces in the container. Note that the first liquid may be fresh water or water added with a chemical.

For example, if the freezing point reducing agent is added, the freezing temperature is lowered. Therefore, in the step of forming the first and second closed spaces in the container, the first liquid to which the freezing point reducing agent is added is used as the first closed space. It is preferable to fill the inside.

In the low-temperature storage method of the present invention, in the step of forming the first and second closed spaces in the container, silicone oil, vegetable oil, gelled Yamanori or gelatin is used as the second liquid as the second liquid. It is preferable to fill the space.

Generally, oils have a large compressibility, and particularly silicone oil has a large compressibility. Therefore, it is preferable to fill the second closed space with silicone oil. In addition, when the object to be stored is food, the safety of food due to liquid leakage can be ensured if the second liquid is vegetable oil having a low freezing point, or gelled Yamatonori or gelatin.

Furthermore, the cryopreservation method of the present invention is not limited to the case where it is applied to pressurized non-freezing cryopreservation in which the storage object is cryopreserved in an unfrozen state, and the preservation object is frozen in a pressurized state to be cryopreserved. Pressurized freezing and cryogenic storage, pressurize the object to be stored in a non-frozen state by pressurization, and then reduce the pressure to form a uniform ice nucleus in the object to be stored to freeze the object to be stored. It can also be applied to laws.

The cryopreservation container of the present invention is a container filled with a first liquid that increases in volume when solidified, is contained in the container, has a higher compressibility than the first liquid, and is lower in temperature than the first liquid. Filled with a second liquid having a freezing point and made of a stretchable inner bag, the temperature between the freezing point of the first liquid and the second liquid, and the atmospheric pressure of the liquid in the storage object The storage object is stored at a storage temperature lower than the freezing point.

The cryopreservation container of the present invention is suitably used in the cryopreservation method of the present invention.

Explanatory drawing which shows the state which concerns on the 1st Embodiment of this invention and cools a cryopreservation container with an iced capsule formation tank. Explanatory drawing which shows a cryopreservation container. Explanatory drawing which shows the cryogenic storage container which concerns on the 2nd Embodiment of this invention.

[First Embodiment]
Hereinafter, a cryopreservation method and a cryopreservation container according to a first embodiment of the present invention will be described with reference to the drawings.

As shown in FIG. 1, the cryopreservation container 10 containing the storage object A is suspended in the contact solvent 4 accommodated in the frozen capsule formation tank 3 by the hanging rod 2 via the hanger 1.

The ice capsule forming tank 3 contains a contact refrigerant 4 such as ethanol brine, and is placed in a freezer compartment (not shown) and cooled. The storage object A is suspended in the contact refrigerant 4 while being stored in the cryogenic storage container 10 shown in FIG.

As shown in FIG. 2, the cryopreservation container 10 has a triple structure, and includes an outer container 11 (container), an inner bag 12, an inner bag 13, and a weight 14, and the upper portion of the outer container 11 is a hanger. 1 is fixed.

The outer container 11 has an upper limit on the internal volume, and is, for example, a bag made of a resin such as a polyethylene film. The inner bag 12 and the inner bag 13 are extendable and made of, for example, a resin such as polyethylene. It is preferable that the inner bag 12 and the inner bag 13 are not in contact with the inner surface of the outer container 11 and the inner surface of the inner bag 12 with a spacer (not shown), respectively.

The inside of the inner bag 13 contains the storage object A and is filled with liquid or gas B. The storage object A is, for example, food such as vegetables, fruits, meat, fish, human or animal organs, cells, etc., and the size and thickness thereof are not particularly limited.

The liquid or gas B filled in the inner bag 13 is not particularly limited, but preferably does not denature the storage object A. For example, the liquid or gas B may be a seasoning liquid such as saline or carbon dioxide gas when the storage object A is a food, or a low-temperature storage liquid when the storage object A is an organ or a cell. Also good.

The inner bag 13 contains the inner bag 13, and the internal space other than the space occupied by the inner bag 4 of the inner bag 12 has a higher compression rate than water (first liquid) and a freezing point lower than that of water. It is filled with the liquid C (2nd liquid) which has.

Liquid C is, for example, oils, gelled Yamatonori or gelatin. In general, oils have a large compressibility, and silicone oil in particular has a large compressibility. Among the silicone oils, dimethyl silicone oil has a large compressibility. Therefore, when the storage temperature is −5 ° C. or lower, the liquid C is preferably a silicone oil, particularly dimethyl silicone oil.

In addition, when the storage object A is a food, if the liquid C is an edible low-freezing point vegetable oil or a gelled Yamatonori, the food safety due to liquid leakage can be ensured. For example, corn oil, coconut oil, and walnut oil have a freezing point at normal pressure of less than 0 ° C., and are suitable when the storage temperature is a relatively high temperature of −2 ° C. to −5 ° C.

The outer container 11 contains the inner bag 12 and the inner space other than the space occupied by the inner bag 12 of the outer container 11 is filled with the liquid D (first liquid) that increases in volume when solidified.

Since the liquid in the storage object A usually occupies most of the liquid, the liquid D is preferably water. However, the liquid D may be fresh water or a solution obtained by adding a drug such as a freezing point lowering agent (freezing inhibitor) to fresh water.

In the cryopreservation method according to the present embodiment, the space in the outer container 11 corresponds to the first closed space in the present invention, the space in the inner bag 12 corresponds to the second closed space in the present invention, and the inner bag. The space in 13 corresponds to the third closed space in the present invention.

Therefore, the cryopreservation method according to the present embodiment forms in the outer container 11 a stretchable first closed space filled with the liquid D and a stretchable second closed space filled with the liquid C. A step of filling the liquid or the gas B to form a third closed space contained in the second closed space, a step of causing the storage object A to be contained in the outer container 11, and a first step. To a third closed space and sealing the outer container 11 in which the storage object A is contained.

In the cryopreservation method according to the present embodiment, the cryopreservation container 10 containing the storage object A is suspended in the contact refrigerant 4 of the freeze capsule formation tank 3, and the freeze capsule formation tank 3 is installed in the freezer compartment.

In the cryopreservation method according to the present embodiment, after the outer container 11 is sealed, the liquid D is cooled to a temperature lower than the freezing point of the liquid D to freeze at least a part of the liquid D, and the internal pressure exceeds atmospheric pressure. In the cryopreservation container 10 raised to a temperature, the temperature is between the step of freezing at least a part of the liquid D to form a frozen capsule and the freezing point of the liquid C and the liquid D, and the object to be stored Storing the storage object A at a storage temperature lower than the freezing point of the liquid (water) in A at atmospheric pressure.

According to the low-temperature storage method according to this embodiment, the liquid D (water) in the outer container 11 is cooled to form an ice capsule. The volume of the inner bag 12 decreases due to volume expansion when water freezes, and the pressure in the inner bag 12 rises to atmospheric pressure or higher.

At this time, the liquid C filled in the inner bag 12 is compressed and its volume is reduced, so that a part of the pressure increase is buffered, and the sudden pressure increase in the inner bag 12 is suppressed, so It is maintained and does not crack. And since the pressure in the inner bag 12 exceeds atmospheric pressure, the freezing temperature of the water in the storage object A is less than 0 ° C., and the storage object A is stored in a non-freezing state at a low temperature of less than 0 ° C. Is possible.

According to the low-temperature storage method according to the present embodiment, since the pressure increase becomes slow, there is no possibility that the outer container 11 is unintentionally damaged, and it is not always necessary to use a pressure resistant container as the outer container 11. Even when a pressure vessel is used as the outer container 11, the wall thickness is small and the pressure resistance may be low, so that a light and inexpensive pressure vessel can be used.

In order to prevent the freezing capsule from breaking at a storage temperature of less than 0 ° C., the volume expansion during freezing of the liquid D (water), the compressibility of the liquid C, and the moisture in the storage object A at the storage temperature The volume ratio between the liquid C and the liquid D may be set in consideration of the pressure that does not freeze.

Thereby, the pressure upper limit value and the pressure increase mode in the outer container 11 are uniquely determined. Therefore, it is possible to stably store the storage object A at a low temperature in a non-freezing state without damaging the outer container 11.

In the conventional freeze pressurization method, for example, as disclosed in Non-Patent Document 1 above, a pressure-resistant container containing a food surrounded by a seasoning liquid is filled with an aqueous solution (seasoning liquid) such as salt or sugar and is -25. By freezing the aqueous solution at 0 ° C., a high pressure of several hundred MPa is applied. In this method, however, the pressure changes when the concentration of the aqueous solution changes slightly, and the food freezes.

On the other hand, in this embodiment, unlike the conventional concentration adjustment, a desired high pressure is obtained by adjusting the amount of liquid, and a desired high pressure can be stably obtained as compared with the conventional case.

This embodiment exhibits a good effect particularly when foods, organs, cells and the like are stored at a low temperature for a relatively short period of time.

Furthermore, if the cryopreservation container 10 that is being cryopreserved in a non-frozen state is immersed in liquid nitrogen according to the present embodiment, the freezing capsule is forcibly generated to depressurize the inside and simultaneously take away the latent heat inside. As a result, it is possible to shorten the freezing time and realize high-quality freezing.

Also, from the relationship between the surface area and the volume, the sphere has the smallest volume for the same surface area. Therefore, when the outer shape of the inner bag 12 filled with the liquid C (water) is a spherical surface, the effect of absorbing the volume increase due to the freezing of the liquid C is small. However, since the sphere can uniformly disperse a large internal pressure, the outer shape of the inner bag 12 should be close to a spherical surface. Therefore, the outer shape of the inner bag 12 is preferably a spheroid. The elliptical flatness is a flatness close to that of a sphere, and is preferably near the limit where the frozen capsule is not broken by the elasticity of the liquid C.

(Examples and Comparative Examples)
[Experiment 1]
First, Experiment 1 was conducted in order to investigate the cryopreservable temperature of the frozen capsule due to the difference in the oil type of liquid C.

In this experiment 1, ethanol brine (contact) at −20 ° C. was used so that the cryopreservation container 10 was not preliminarily cooled in ice water without containing the storage object A and then touched the bottom wall and side wall of the freeze capsule formation tank 3. Refrigerant) 4 was immersed in a suspended state, and fresh water (liquid D) in outer container 11 was frozen to form a frozen capsule.

As the outer container 11, a bag made of polyethylene film having a clamping chuck at the top was used. This bag had a length under the chuck of 140 mm, a width of 100 mm, and a thickness of 0.04 mm. As the inner bag 12, a bag made of a polyethylene film having a fastening chuck on the upper side was used. This bag had a length under the chuck of 85 mm, a width of 60 mm, and a thickness of 0.04 mm.

Since it was frozen in ethanol brine at −20 ° C., the cooling rate was fast, and the water was frozen from the outside over the entire inner surface of the outer container 11 and frozen in about 5 minutes. When frozen, the refractive index changed and the inside became dark.

In Example 1 and Comparative Examples 1 to 3, edible walnut oil was used as liquid C.

In Example 1 and Comparative Examples 1 to 3, frozen capsules were formed using a volume ratio of 7 for water and 9 for edible walnut oil. The frozen capsules were stored in a refrigerator at low temperatures for 3 days at storage temperatures of −3 ° C., −7 ° C., −18 ° C., and −60 ° C. The control temperature range of the storage temperature is 2 ° C. above and below the center temperature.

In Example 2 and Comparative Examples 4 and 5, the same treatment as in Example 1 and Comparative Examples 1 to 3 was performed using dimethyl silicone oil having a viscosity of 10 mm / s as liquid C. The experimental results are shown in Table 1.

As can be seen from Table 1, when liquid C was edible walnut oil, no cracking occurred in the frozen capsule at −3 ° C. (Example 1), but no crack occurred in the frozen capsule at −7 ° C. or less (comparison). Examples 2 to 3).

On the other hand, when the liquid C is dimethylsilicone oil and the volume ratio of dimethylsilicone oil to water is 9: 7, the frozen capsules did not crack when the storage temperature was -7 ° C or higher (Example 3). When the storage temperature was −18 ° C. or lower, the frozen capsule was cracked (Comparative Examples 4 and 5).

As described above, when the liquid C is dimethyl silicone oil, the storage temperature at which the frozen capsule does not crack is lower than when the liquid C is edible walnut oil. This is presumably because dimethyl silicone oil has a higher compressibility than edible walnut oil and can absorb more volume expansion due to freezing of liquid D (water).

Furthermore, when the liquid C was dimethyl silicone oil, cracking occurred in the frozen capsule when the storage temperature was −18 ° C. or less when the volume ratio of dimethyl silicone oil to water was 9: 7 (Comparative Example 4).

[Experiment 2]
Next, frozen capsules were formed by the same method and conditions as in Experiment 1, and Experiment 2 was performed at the same low temperature storage conditions as in Experiment 1. In Experiment 2, 10 ml of 1% saline solution as liquid B, 90 ml of dimethyl silicone oil having a viscosity of 10 mm / s as liquid C, 70 ml of fresh water as liquid D, and 1.5% agar as storage object A were used. The results of Experiment 2 are shown in Table 2. Comparative Example 7 is a control sample at 20 ° C. that is not stored at low temperature.

The 1.5% agar used as the storage object A is a sample formed by mixing a Takano tofu powder in a 1.5% by weight agar aqueous solution. The moisture content of this sample is about 80%, which is close to the moisture content of meat such as seafood and animal meat, which is 65% to 85%.

This sample was specifically prepared as follows. First, Koya tofu was crushed with a grater and powdered. Then, 7% by weight of Takano tofu powder with respect to the weight of water was mixed with the boiled 1.5% by weight agar aqueous solution, stirred for 5 minutes, placed in the container, and cooled with ice water to solidify the contents. . And after removing the condensed water droplets in the container, die-cutting was performed using a cylindrical mold having an inner diameter of 12 mm to obtain a cylindrical sample having a diameter of 12 mm and a height of 10 mm. Further, the molded sample was stored in a plastic bag with a chuck and refrigerated at 4 ° C. for 1 day.

The drip rate R was determined using this sample as follows. First, after measuring the weight Wp of the sample before cryopreservation, the soot sample was cryopreserved. Then, the sample was placed in a centrifuge tube (Spitz tube) in a cryopreserved state, and naturally thawed by centrifuging at 113 G for 5 minutes using a swing rotor centrifuge. And the sample was taken out from the centrifuge tube, the weight Wc was measured, and the drip rate R was calculated | required by Formula (1) based on the weight difference of a sample.

R = 100 × (Wp−Wc) / Wp (1)
However, since there was variation in the production of samples, the drip rate R was compared using samples of the same production lot.

In addition, since the outer container 11, the inner bag 12, and the inner bag 13 were all transparent, it was visually determined whether the seasoning water and the sample were frozen or not frozen. The experimental results are shown in Table 1.

In Examples 5 and 6, the drip rate R was almost the same as that of Comparative Example 7 which was not stored, and the storage state was good visually. Therefore, it was found that the product could be well stored for 3 days in a non-frozen state at a low temperature of −7 ° C.

This is considered to be because the sample was successfully stored at a low temperature in a non-frozen state by slowing the pressure rise due to the presence of dimethyl silicone oil.

Moreover, in Example 7, although the freezing capsule cracked, the drip rate R was small as compared with Comparative Example 6 without dimethyl silicone oil. This is presumably because in Example 7, the frozen capsules were cracked after deep supercooling once, so that the sample tissue was not damaged much.

As can be seen from Example 7, even when cryopreserved in a frozen state, the presence of dimethylsilicone oil takes a long time until ice breaks, and the sample is not frozen due to a large freezing point depression due to pressurization. Since the ice nuclei are uniformly formed inside the sample by freezing due to the ice breakage from the state, the sample is frozen and can be stored at a low temperature of good quality.

As described above, from the results of Experiment 2, it was found that the presence of dimethylsilicone oil alleviated the rapid pressure increase caused by freezing, and the internal pressure could be increased while preventing the freezing capsule from cracking.

[Second Embodiment]
Hereinafter, a low temperature storage method and a low temperature storage container according to a second embodiment of the present invention will be described with reference to the drawings.

As shown in FIG. 3, the cryopreservation container 20 includes an outer container 21, a first inner bag 22, and a second bag 23. Although not shown, the upper portion of the outer container 21 is the hanger 1 (see FIG. 1). Fixed to.

The outer container 21 is, for example, a container made of a resin such as polyethylene terephthalate (PET) or a metal such as iron. The first bag 22 and the second inner bag 23 are extendable and made of, for example, a resin such as polyethylene.

The first bag 22 is filled with the liquid C having a compression rate larger than that of the liquid D and having a freezing point lower than the freezing point of the liquid D in the first closed space inside. The liquid C filled in the first bag 22 is oils, gelled Yamatonori, gelatin, and the like, similar to the liquid C filled in the inner bag 22.

The second bag 23 is filled with the liquid D whose volume increases when solidified in the second closed space inside. Here, the liquid D is water. However, the liquid D may be fresh water or a solution obtained by adding a drug such as a freezing point lowering agent to fresh water.

The outer container 21 contains the first bag 22, the second bag 23, and the storage object A, and the inner space other than the space occupied by these in the outer container 21 is filled with an antifreeze E such as an ethanol aqueous solution. The

The storage object A is, for example, food such as vegetables, fruits, meat, fish, human or animal organs, cells, and the like, and the size and thickness are not particularly limited, but are preferably smaller.

In this embodiment, similarly to the first embodiment, the cryogenic storage container 20 containing the storage object A is cooled to a storage temperature in a freezing chamber (not shown).

According to this embodiment, the liquid D (water) in the second bag 23 is cooled and frozen. Due to the volume expansion during freezing, the volume of the liquid portion in the outer container 21 decreases, and the pressure in the outer container 21 rises above atmospheric pressure.

At this time, a part of the pressure rise is buffered by the compression of the liquid C filled in the first bag 22, and a sudden pressure rise in the outer container 21 is suppressed. And since the internal pressure of the antifreeze liquid E exceeds atmospheric pressure, the freezing temperature of the water in the preservation | save object A will be less than 0 degreeC, and it is possible to preserve | save the preservation | save object A in a non-freezing state at the low temperature of less than 0 degreeC. Become.

According to the present embodiment, the outer container 21 is not likely to be inadvertently damaged by an excessively high pressure, and the wall thickness is thin and the pressure resistance may be low. Therefore, a lightweight and inexpensive pressure container can be used. .

In order to prevent the outer container 21 from being damaged even at a storage temperature of less than 0 ° C., the volume expansion during freezing of the liquid D (water), the compressibility of the liquid C, and the moisture in the storage object A at the storage temperature. The volume ratio between the liquid C and the liquid D may be set in consideration of the pressure at which the liquid does not freeze.

Thereby, the upper limit value and the rising mode of the pressure applied in the outer container 21 are uniquely determined. Therefore, it is possible to stably store the storage object A at a low temperature in a non-freezing state without damaging the outer container 21.

Unlike the conventional concentration adjustment, the present embodiment obtains a desired high pressure by adjusting the amount of liquid, and can stably obtain a desired high pressure as compared with the conventional case.

As mentioned above, although embodiment of this invention was described, this invention is not limited to this. For example, the case where the

cryopreservation containers

10 and 20 are suspended and cooled in the contact refrigerant 4 of the ice capsule forming tank 3 has been described. However, the apparatus and method for cooling the

cryopreservation containers

10 and 20 are not limited to this.

In deep sea surveys, etc., deep sea creatures are stored together with seawater in a pressure vessel in the deep sea, and a bag containing a buffer solution such as dimethyl silicone oil is put in to reduce the sudden decompression speed when ascending. Damage to marine life can be reduced. If the inside of the pressure vessel is only seawater, if the external pressure is reduced during the ascent, the water has a low compressibility and a slight increase in volume due to the reduced pressure, so that the pressure vessel is deformed and it becomes difficult to maintain the pressure. If a liquid with a large compressibility, such as dimethyl silicone oil, is placed in the pressure vessel, the volume change is much larger than the volume increase due to deformation of the pressure vessel during decompression, so it is possible to ease the decompression. .

Claims

A second, expandable, closed space filled with a first liquid that increases in volume upon solidification, and a second freezing point that has a higher compressibility than the first liquid and a lower freezing point than the first liquid. Forming a telescopic second closed space filled with liquid in the container;
A step of storing the object to be stored in the container;
Sealing the container in which the first and second closed spaces are formed and the object to be stored is contained;
After sealing the container, the first liquid is cooled to a temperature lower than the freezing point of the first liquid, the temperature is between the freezing points of the first liquid and the second liquid, and in the storage object. And a step of storing an object to be stored at a storage temperature lower than the freezing point of the liquid at atmospheric pressure.
In the step of forming the first and second closed spaces in the container, the second closed space is formed in the first closed space;
In the step of storing the storage object in the container, the storage object is stored in the second closed space;
In the step of increasing the pressure in the container, at least a part of the first liquid is frozen to generate an ice capsule, and the pressure in the second closed space is increased to a pressure exceeding atmospheric pressure. The low-temperature storage method as described.
In the step of forming the first and second closed spaces in the container, the volume ratio between the first liquid and the second liquid is set so that the frozen capsule is maintained in the step of storing the storage object. The low-temperature storage method according to claim 2.
The cryopreservation method according to claim 2 or 3, further comprising a step of forming a third enclosed space filled with a liquid or a gas and existing in the second enclosed space.
5. The cryopreservation method according to claim 1, wherein in the step of forming the first and second closed spaces in the container, the first closed space is filled with water as the first liquid. 6.
6. The step of forming the first and second closed spaces in the container fills the second closed space with silicone oil, vegetable oil, gel-like Yamanori or gelatin as the second liquid. The low-temperature storage method according to any one of the above.
7. The method according to claim 1, wherein in the step of forming the first and second closed spaces in the container, the first liquid to which the freezing point reducing agent is added is filled in the first closed space. Low temperature storage method.
A container filled with a first liquid that increases in volume when solidified;
The container is filled with a second liquid having a compressibility higher than that of the first liquid and having a freezing point lower than that of the first liquid.
The storage object is stored at a storage temperature that is between the freezing point of the first liquid and the second liquid and lower than the freezing point of the liquid in the storage object at atmospheric pressure. Cryogenic storage container.