JP2007289157A - Cryopreservation method for cell or tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique suppressing impairment of cells or tissues without using additives such as a cryoprotective agent, etc., and imparting a high quality cryopreservation. <P>SOLUTION: The cryopreservation method is characterized by comprising a drying process drying the cells or tissues at normal temperature to an extent not impairing the cells or tissues before cryopreservation of the cells or tissues, and then subjecting the dried cells or tissues to the cryopreservation. The preferable method freezes the cells or tissues at the temperature below the maximum ice generation temperature range, and in drying at normal temperature a reduced-pressure microwave heating is used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method for cryopreserving cells or tissues, specifically food or living organisms.

In cryopreservation of foods and living organisms, it is required from the viewpoint of long-term preservation of high-quality foods and transplantation treatment to prevent cell damage and perform cryopreservation while remaining as alive as possible. In cryopreservation of food, living bodies, etc., the most important point in preserving quality and function is how quickly a temperature region called a maximum ice crystal formation zone is passed through and cooled. In this temperature zone, ice crystals are easily generated and grow. Therefore, if the temperature crystal is in this temperature zone for a long time, the ice crystals grow and become large ice crystals, and the cells and tissues are damaged.

For this reason, it has been devised that rapid freezing is allowed to pass through this temperature zone in a short time, or that the ice is frozen as fine ice crystals at a lower temperature without being frozen in this temperature zone. In the former case, a powerful refrigerator is used or heat transfer is promoted. However, there are problems in that a large amount of energy is required and the cooling capacity is limited. On the other hand, in the latter, the formation of large ice crystals is prevented by using a frost damage protective agent such as glycerin (see Patent Document 1), and recently, the maximum ice crystal temperature zone is not frozen by applying a magnetic field and an electric field. There has been proposed a method (see Patent Document 2) in which, after passing through cooling as it is, it freezes instantaneously to form fine ice crystals and freezes without damage. However, the use of anti-frost damage agents is not suitable for food. In addition, the influence of the electromagnetic field is still unclear, and there is still no confirmation of its effectiveness.
JP-A-6-292564 JP 2001-245645 A

Because of this technical background, it is possible to pass through the maximum ice crystal zone unfrozen even at a slow cooling rate without using frost protection agents or additives, resulting in finer ice crystals at lower temperatures and damage to cells and tissues. Development of a method that can be cryopreserved in a state that does not give odor is desired.

An object of the present invention is to provide a cryopreservation technique capable of suppressing cell or tissue damage. Another object of the present invention is to provide a technique that enables high-quality cryopreservation without using additives such as frost damage prevention agents.

The present inventor has found that the viability of cells during cryopreservation is greatly influenced by intracellular freezing and extracellular freezing, and the number of water molecules involved in freezing is reduced by drying at room temperature in advance. And a cryopreservation method for preventing cell damage due to ice crystal formation.

That is, in the invention described in claim 1 of the present invention, when cryopreserving a cell or tissue, the cell or tissue is first dried to an extent that does not damage the cell or tissue at room temperature, and then frozen. It is a cryopreservation method for cells or tissues characterized by preservation.

The invention described in claim 2 of the present invention is the cell or tissue cryopreservation method according to claim 1, wherein the cell or tissue is frozen at a temperature below the maximum ice crystal formation zone.

The invention described in claim 3 of the present invention is the method for cryopreserving cells or tissues according to claim 1 or 2, characterized in that microwave vacuum drying is used as room temperature drying.

And the invention described in claim 4 of the present invention does not add an additive such as a frost damage preventive agent in the case of frozen storage, and the cell or tissue according to any one of claims 1 to 3 This is a cryopreservation method.

In addition, for example, in JP-A-10-127263, as a drying method that preserves the flavor of food and can be stored for a long time, the material to be dried is kept until the moisture in the material to be dried becomes 10 to 30% of the moisture in the fresh state. A method of pre-drying at a non-freezing temperature, for example, 10 to 40 ° C., followed by vacuum freeze-drying is disclosed. However, the present invention relates to vacuum freeze-drying and is completely different from the present invention aimed at cryopreservation without damaging cells and tissues.

According to the cryopreservation method of the present invention, in the case of foods, high-quality long-term frozen storage with little drip when thawed is possible. Further, in the medical field, long-term cryopreservation is possible in which there is little damage to living cells or living tissues and no addition of a frost damage protective agent or the like is required.

In the present invention, when a cell or tissue is cryopreserved, the cell or tissue is first dried to an extent that does not damage the cell or tissue at room temperature, and then frozen and stored. In the present invention, normal temperature means a temperature range of about 10 to about 30 ° C., and the dryness that does not damage cells or tissues can be determined by the moisture content of the cells or tissues, for example, onion cells In this case, the moisture content on the dry basis may be dried to about 4 g / (g · dry). This moisture content can be easily determined by observing the object under a microscope, for example.

In cryopreservation of food, living bodies, etc., the most important point in preserving quality and function is how quickly a temperature region called a maximum ice crystal formation zone is passed through and cooled. In this temperature zone, ice crystals are easily generated and grow. Therefore, if the temperature crystal is in this temperature zone for a long time, the ice crystals grow and become large ice crystals, and the cells and tissues are damaged. The temperature of the maximum ice crystal formation zone is generally said to be 0 ° C. to −5 to −7 ° C., but the temperature zone varies depending on the type of cells and tissues. In the present invention, it is preferable to freeze at a temperature below this maximum ice crystal formation zone.

In the present invention, the method and means for drying to the extent that does not damage cells or tissues at room temperature are not particularly limited, but it is preferable to use microwave vacuum drying as room temperature drying. Microwave vacuum drying is a vacuum drying method in which an object such as a cell or tissue is placed in a chamber connected to a vacuum pump, and the object is irradiated with microwaves in a vacuum state. Among microwave drying under reduced pressure, the method and apparatus proposed by the present inventors in the international application WO2005 / 100891 is preferable. This method is a microwave vacuum drying method, in which a) the degree of decompression in the chamber is set to a saturated vapor pressure or less corresponding to the alteration temperature of the object, and b) a gas such as air is supplied into the chamber from the outside of the chamber. And c) a method of performing drying in a state in which the microwave is turned on and off and the temperature of the object is kept below the alteration temperature. Using this microwave vacuum drying, cells or tissues can be uniformly dried at room temperature to an optimal moisture content. Thereafter, cryopreservation is performed, but it is desirable that the cooling rate be as high as possible.

In the present invention, particularly when foods are targeted, it is preferable not to add an additive such as a frost damage protective agent during frozen storage.

In the present invention, the cell or tissue is dehydrated by pre-drying prior to freezing, and the number of water molecules involved in freezing is reduced to allow passage through the maximum ice crystal formation zone. More specifically, using a microwave vacuum drying method that can be uniformly and dried at room temperature, the cells are dried to a minimum moisture content that can maintain high cell viability or maintain good cell conditions. Thereby, in the subsequent freezing process, the intracellular freezing temperature is lowered, so that fine ice crystals with little damage to the cells are generated, and the growth of ice crystals during the storage period can be suppressed. As a result, cell or tissue damage is reduced by the fine ice crystals, and the preservation of living tissue is improved. In the present invention, the method and means for freezing and storing are not particularly limited, and known methods and means can be used. Hereinafter, the present invention will be described in detail by way of examples.

First, an onion epidermis tissue in which the freezing behavior of cells can be observed was dried at room temperature and then frozen with a microscope equipped with a cooling stage, and the temperature at which intracellular freezing occurred and the state of ice crystals were observed. The onion epidermal tissue used in the experiment was subjected to life / death determination with trypan blue staining solution, and after confirming that the cells were in a viable state, the cells were left at room temperature and air-dried. By changing the standing time, the degree of drying was changed and the water content was changed. In addition, viability was determined with trypan blue, and the relationship between moisture content and survival rate was determined. The results are shown in FIG. It was considered meaningless if the cells died due to drying, and subsequent freezing experiments were performed on cells with a moisture content in the survival range.

The cooling rate of the onion epidermal tissue in the freezing experiment was set to a slow rate of about 1 ° C./min in order to see the effect of dry dehydration. The relationship between the temperature at which intracellular freezing occurs and the water content is shown in FIG. In the case of onion cells, even if they are not dried, some freeze at a temperature lower than the maximum ice crystal formation zone, which is about 0 ° C to -7 ° C. It turns out that it freezes at a temperature lower than the maximum ice crystal formation zone, and the freezing temperature decreases with drying. From this, it was confirmed that the cells dried in advance were intracellular frozen at a lower temperature and could pass through the damaging maximum ice crystal formation zone in an unfrozen state.

Observation of cells during freezing with a digital microscope using transmitted illumination (not shown), frozen cells become dark, but cells with high water content have good light transmission and are not so dark. On the other hand, in the cells that had been dried to reduce the water content, fine ice crystals were formed, and light refraction advanced, resulting in dark images. From this, it can be judged that pre-drying is effective for cryopreservation because it produces fine ice crystals that do not damage cells.

Next, in order to show that the present invention is an effective method in an actual freezing operation, a freezing experiment of raw oysters was performed and the amount of drip after thawing was compared. The results are shown in FIG. Using 15-17 g of raw oysters, it was thawed after freezing and the amount of drip at that time was compared. For drying before freezing, microwave vacuum drying was used (see WO 2005/100891), and the drying temperature was 30 ° C. For freezing, the freezer room of a household refrigerator was used. As can be seen from the results in FIG. 3, it was confirmed that the amount of drip after thawing was reduced by prior room temperature drying, and cell damage was suppressed. From the above, it was shown that the present invention is an effective method for cryopreservation.

After pre-drying with cocoons and cocoons (2 cm square) to a constant moisture content as in Example 2, the cocoons were frozen at −20 ° C. and cocoons were frozen at −20 ° C. or −80 ° C. And stored frozen in a -20 ° C freezer. What was stored as it was for one week was thawed with running water. And the sensory test was done about four items, a taste, smell, a color, and food texture, by the panelist. All the items were raw, and the sardines were scented and colored, and the taste and texture were steamed.

The nephew was carried out by 12 panelists and the nephew was carried out by 11 panelists. First, eat a fresh sample, memorize the taste and smell, and set it as the reference 0 point. Then, it is evaluated based on the evaluation score table of Table 1 how other various samples were compared with this. Regarding items other than the taste, referring to Table 1, a low score was given to a fresh one. As shown in Table 2, the results obtained by pre-drying about 3% were highly evaluated in both cases. In addition, the higher the cooling rate ((b) in Table 2) of the soot was evaluated highly. In the case of firewood, the slower cooling rate (Table 2 (a)) was cooled to −20 ° C., and the faster cooling rate (Table 2 (b)) was cooled to −80 ° C. Is.

Various samples were pre-dried before freezing in the same manner as in Example 2 to examine changes in the drip rate (drip amount) generated after thawing. For raw oysters, the amount of protein contained in the drip was measured, and the relationship with the degree of cell tissue destruction was examined. Here, the drip rate is the ratio of the drip amount to the mass before thawing when the mass difference between the mass before thawing and the mass after thawing is defined as the drip amount. As samples, 100 g of raw oysters, 100 g of strawberries, 100 g of strawberries, 100 g of strawberries, 130 g of figs, 45 g of chicken fillets, and 55 g of boiled eggs were used.

FIG. 4 shows the drip rate of raw oysters, and FIG. 5 shows the ratio of protein contained in 100 g of raw oysters. The moisture content of raw oysters is 5.8 g (not shown) per gram of dry oysters, 5.4 g of dried oysters by 5% of the total mass, and 10% dried similarly. Was 5.07 g and 15% dried was 4.75 g. Since the amount of drip decreased by pre-drying and the protein in the drip also decreased, it was confirmed that pre-drying before freezing can suppress cell damage. It can also be seen that the greater the cooling rate, the lower the drip and cell damage.

Pre-dried persimmon (Fig. 6), persimmon (Fig. 7), strawberry (Fig. 8), fig (Fig. 9), chicken fillet (Fig. 10) and boiled egg (Fig. 11) under the same conditions as for raw oysters The amount of drip when cooled in the same manner is shown in FIGS. In any case, the drip can be reduced by the reduction of the moisture content due to the preliminary drying.

4-11, “Refrigerator” means that it was cooled to −20 ° C. and frozen in that atmosphere, and “Cryogenic freezing” means that it was cooled to −80 ° C. and frozen in that atmosphere. means. Those not described are those cooled to −20 ° C. and frozen in that atmosphere. All the storages are frozen at -20 ° C.

The tissue changes due to drying and freezing were observed. The frozen section was cut to a thickness of 10 μm with a microtome, stained with a hematoxylin / eosin solution, and then observed with a digital microscope. The magnification was 300 times. FIG. 12 shows the observation result in the case of a heel. Comparing the state of the tissue before freezing and after thawing, the one that was frozen without pre-drying had large cavities due to ice crystals in the cells, whereas the one that had been pre-dried had cavities inside the cells. It can be seen that it is decreasing or decreasing. This indicates that the pre-dried tissue is less damaged than the tissue that was not pre-dried and can be frozen while maintaining its shape.

As shown in FIG. 13, even in the case of boiled eggs, the traces of ice crystals that cause cell damage are reduced along with preliminary drying, indicating that preliminary drying is a cryopreservation method with little damage. It was.

From the above sensory test and tissue observation, it was proved that the food should be cryopreserved after preliminary drying to a moisture content of about 3 to 5%. Moreover, since the amount of drip fell with predrying and the protein mass which flows out also decreased, it turned out that cell destruction is suppressed by predrying. This shows that the present invention is also effective for maintaining the quality of foodstuffs.

The present invention is useful, for example, for freezing and cryopreservation of foods, or cryopreservation of living organisms or living tissues, and the latter can be used in fields such as transplantation therapy, breed improvement, and new drug development.

It is a figure which shows the relationship between the moisture content of an onion cell, and a survival rate. It is a figure which shows the relationship between the moisture content of an onion cell, and intracellular freezing temperature. It is a figure which shows the relationship between the drying time at the normal temperature of raw oyster, and the drip amount. It is a figure which shows the relationship between the drip rate of a raw oyster, and a moisture content. It is a figure which shows the ratio of the protein contained in the drip of a raw oyster. It is a figure which shows the relationship between the drip rate of a cocoon, and a moisture content. It is a figure which shows the relationship between the drip rate of a cocoon, and a moisture content. It is a figure which shows the relationship between the drip rate of a strawberry, and a moisture content. It is a figure which shows the relationship between the drip rate and the moisture content of FIG. It is a figure which shows the relationship between the drip rate and moisture content of chicken fillet. It is a figure which shows the relationship between the drip rate of a boiled egg, and a moisture content. It is a figure which shows the structure | tissue change of a heel. It is a figure which shows the structure | tissue change of a boiled egg.

Claims (4)

A method for cryopreserving a cell or tissue, wherein the cell or tissue is first cryopreserved at room temperature so that the cell or tissue is not damaged, and then frozen and stored. 2. The method for cryopreserving cells or tissues according to claim 1, wherein the cells are frozen at a temperature below the maximum ice crystal formation zone. 3. The method for cryopreserving cells or tissues according to claim 1 or 2, wherein microwave drying under reduced pressure is used as room temperature drying. The cryopreservation method for cells or tissues according to any one of claims 1 to 3, wherein an additive such as a frost damage preventive agent is not added during cryopreservation.

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