WO2010008059A1

WO2010008059A1 - Method for storing cells and device therefor

Info

Publication number: WO2010008059A1
Application number: PCT/JP2009/062918
Authority: WO
Inventors: 昭夫清水; 進一早乙女; 一行中嶋
Original assignee: 学校法人創価大学; タマティーエルオー株式会社
Priority date: 2008-07-18
Filing date: 2009-07-16
Publication date: 2010-01-21
Also published as: JP2010022269A

Abstract

Provided are a method for storing cells whereby the requirements for the quickness and convenience in treating the cells before and after the storage and for the easiness in transporting the same can be satisfied while maintaining the survival rate of the cells at a high level, and a device therefor. A method for storing cells wherein the storage temperature is controlled to be higher than the crystallization temperature of water in the cells to be stored and not higher than the optimum action temperature of the cells while the storage pressure is controlled to 0.11 to 10 MPa. A device for storing cells comprising a pressurized container which can be maintained under a definite pressure when packed with a pressure medium, and one or more cell storage containers which can be put in the pressurized container and have an ability to transfer the pressure from outside to a material contained therein so that cells enclosed therein can be pressurized.

Description

Cell storage method and apparatus

The present invention relates to a cell storage method and an apparatus thereof, and more particularly to an improvement of a storage method for storing cells without freezing and an apparatus thereof.

Cells collected from living organisms can be cultured in vitro to examine their physiological activities and functions, used as samples for pharmaceutical development, and used for treatments such as transplantation. Yes. Particularly in recent years, due to social criticism of animal experiments, it has become an international trend to minimize animal experiments and replace them with cultured cells. There are two types of cell culture: primary culture that maintains a high level of cell properties in vivo, and subculture for making a cell line with certain stable properties. Of these, primary cell cultures are increasingly being used as alternatives to the animal experiments described above. To that end, many different experiments have been performed while maintaining the properties in vivo, and comparison experiments under the same conditions have been performed. Therefore, it is required that the cells can be stored and transported easily with a good survival rate. In addition, there is a high demand for a quick and simple storage method for other cells.

As a general storage method of cells, there is frozen storage, but this method cannot satisfy the above-mentioned requirement of “rapid and simple”. In recent years, a method for storing food and microorganisms under high pressure of 500 to 10000 atm (about 50 to 1000 MPa) in order to store them without freezing has been disclosed (Patent Document 1). In addition, in the storage of biological substances such as blood, platelets, and heart, a storage method is disclosed under a low temperature and a high pressure of 70 to 1000 atmospheres (about 7 to 100 MPa) in combination with a storage solution (Patent Literature). 2). All of these are technical ideas of applying as low pressure as possible and non-freezing, and thus applying ultra-high pressure.

Therefore, these storage states are extremely supercooled, and when transporting the preserved material, the balance of the supercooled state is lost due to physical shock during transportation, and moisture in the preserved material freezes at a stretch. There is a risk that the stored material will be damaged. Further, the prior art is not a technique for storing the cells themselves under pressure.

Based on the above circumstances, the inconvenience of the existing storage method is accepted and used for the experiment, or when preserving in vivo properties is the top priority, cells are collected from living organisms every time the experiment is conducted. is there.

Japanese Patent No. 2533584 Special Table 2002-528469

Accordingly, an object of the present invention is to provide a cell storage method and apparatus capable of meeting the demands of quickness, simplicity, and ease of transportation before and after cell storage while maintaining good cell viability. It is to provide. Further, the purpose of the present invention is to provide several types of experiments when it is desired to carry out many various experiments while maintaining the properties of cells in vivo or during culture, and when it is desired to quickly conduct comparative experiments under the same conditions. It is also included to provide a storage method and apparatus capable of storing the cells at the same time under the same conditions.

In order to solve the above-mentioned problems, the present inventors have conducted extensive research. As a result, by placing the cells under a certain pressure, the temperature of the water is higher than the crystallization temperature of the intracellular water, but the activity of the cells is low. The inventors have found that cells can be stored with a high survival rate if the temperature is not more than the optimum temperature, and the present invention has been completed.

That is, in the cell storage method of the first aspect of the present invention, the storage temperature is higher than the crystallization temperature of intracellular water to be stored, the cell activity optimum temperature or less, and the storage pressure is 0.11 to This is a storage method of 10 MPa. Here, the optimum temperature of the cell activity means a temperature suitable for the life activity of the cell, and the temperature varies depending on the cell type. For example, in the case of an animal cell, the body temperature of the animal is That is true.

Preferably, the storage temperature is 1 ° C. to 37 ° C., and the storage pressure is 0.11 to 6.5 MPa.

Preferably, the cells are seeded or suspended in a medium that is liquid under the aforementioned storage conditions.

Preferably, the cells are sealed in a storage container made of a material that can transmit external pressure to the internal substance, and the cells are pressurized through the storage container with a pressure medium.

Preferably, the method for storing cells includes a step of enclosing cells in a storage container having a material capable of transmitting external pressure to an internal substance, and one or more of the storages including the cells. Storing the container in a pressurized container; maintaining the pressure container in the temperature; and maintaining the pressure by filling the pressurized container with a pressure medium; And pressurizing the cells.

Preferably, the pressure medium is a liquid.

The cell storage device according to the second aspect of the present invention includes a pressurized container capable of maintaining pressure by filling with a pressure medium, and can be accommodated in the pressurized container. One or two or more cell storage containers capable of pressurizing cells enclosed inside by having a material that can be transmitted to a substance.

Preferably, the cell storage device adjusts the pressure in the pressurized container by sending the pressure medium into the pressurized container by a fluid transfer device.

Preferably, in the cell storage device, the pressure medium is a liquid.

Preferably, in the cell storage device, the pressure increase / decrease rate can be adjusted by controlling the feeding and discharging of the pressure medium by the fluid transfer device.

According to the present invention, cells can be preserved in response to demands for quickness, simplicity, and ease of transportation before and after cell preservation while maintaining good cell viability. Furthermore, according to the present invention, several types of cells can be used once when a large number of various experiments are performed while maintaining the properties in vivo or during culture, or when a comparative experiment under the same conditions is performed quickly. Can be stored under the same conditions.

It is a figure which shows the whole schematic of an example of the cell storage apparatus of this invention. It is the state which stored the storage container in the pressurized container, Comprising: Sectional drawing of both containers is represented roughly.

Specific embodiments of the present invention will be described in detail below. Cells that can be stored in the present invention are not particularly limited and can be applied to storage of animal cells, microbial cells, etc., but animal cells are preferable for the purpose of storage, for example, mice, rats, guinea pigs, birds, dogs, cats, pigs It can be applied to the preservation of various cells collected from animals such as sheep, horses, cows, monkeys, and humans. As cell types, it can be applied to brain cells, nerve cells, epidermal cells, fiber cells, muscle cells, hepatocytes, stem cells, etc. It can also be applied to embryonic stem cells and artificial pluripotent stem cells it is conceivable that.

In the preservation method of the present invention, the cells are adhered to the container wall of a cell culture container such as a plate or flask, or the flask is filled with a culture solution after the adhesion, or the cells are placed in a suitable container. The cells can be stored in a state inoculated in a solid or liquid medium or suspended in a liquid medium in a suitable container. From the viewpoint of uniformly applying pressure to the cells, the cells are preferably stored in a state of being suspended in a liquid medium. As the liquid medium, for example, Dulbecco's Modified Eagle Medium (commonly called “DMEM”) or the like is used. Hereinafter, the container for storing the cells is referred to as “storage container”.

As a method for applying pressure to cells, the storage container is placed in a container for applying pressure (hereinafter referred to as “pressurized container”), and a gas or liquid is used as a pressure medium in the storage container. Apply pressure to the cells. The number of storage containers stored in the pressurized container may be one, or two or more may be used when several types of cells are desired to be stored at the same time. The number of storage containers stored in the pressurized container is preferably 100 or less from the design of the apparatus.

Here, the pressure medium is a substance for applying a predetermined pressure directly or indirectly to the cells in the storage container and maintaining the pressure, and is a gas or liquid. As the gas, air, oxygen, carbon dioxide, nitrogen, a rare gas, or a mixed gas thereof can be used. The gas is appropriately selected depending on the cell type and storage period. Although there is no restriction | limiting in particular as a liquid, Water, methanol, ethanol, ethylene glycol, glycerin, antifreeze liquid, inorganic salt aqueous solution, such as salt or sodium sulfate, aqueous solution of saccharides, these mixtures etc. can be used. Water is preferable in terms of simplicity and cost.

The first method in the case of using a gas as the pressure medium applies pressure by putting the storage container into a pressurized container in an open state. In this case, the pressurized container may not be used, and the inside of the storage container may be directly pressurized with the gas and then sealed to be in a pressurized state. The material of the storage container is not particularly limited as long as it can withstand the applied pressure. For example, glass, stainless steel, or a resin product such as polyethylene, polypropylene, acrylic resin, silicon resin, or Teflon (registered trademark) is used. The pressure is appropriately selected according to the magnitude of the applied pressure.

The second method in the case of using a gas as the pressure medium is similar to the case of using a liquid as the pressure medium described below, and uses a material that can transmit external pressure to the internal substance in order to apply a predetermined pressure to the cells. The storage container is used, and the storage container is sealed and placed in a pressurized container.

When a liquid is used as the pressure medium, the storage container needs to be sealed and placed in a pressurized container in order to prevent cells from coming into contact with the liquid. Therefore, in this case, in order to apply a predetermined pressure to the cells, a storage container having a material capable of transmitting an external pressure to an internal substance is used. There is no particular limitation as long as this requirement is satisfied, but for example, synthetic resin such as silicon resin, natural rubber, SBR, synthetic resin such as vinyl chloride resin, and Teflon (registered trademark) can be used. . In this case, it is desirable that the cells are suspended in a liquid medium and sealed in the storage container so that the pressure is accurately transmitted to the cells in the storage container.

In the second method in the case of using a gas as the pressure medium and in the case of using a liquid, the cells are sealed in a storage container together with a medium appropriately selected according to the storage period, etc. The pressure medium does not need to be changed with the cell type because it is not touched.

The shape of the storage container may be any shape as long as pressure is transmitted to the inside, for example, a spherical shape, a cylindrical shape, or a bag shape such as an infusion bag.

The cells placed in the storage container as described above are stored at a temperature higher than the crystallization temperature of the water in the cells, not more than the optimum temperature for cell activity, and a pressure of 0.11 to 10 MPa. The water crystallization temperature varies depending on the cell type, but is generally 0 to -10 ° C. If the temperature is lower than the crystallization temperature, the water in the cell may crystallize and destroy the cell.If the temperature is higher than the optimum temperature for the activity, it is generally an unsuitable environment for the cell and the cell viability decreases. is there. This is because when the pressure is lower than 0.11 MPa, no significant difference is observed as compared with the case of storage at atmospheric pressure, and the cell viability is low, and when it exceeds 10 MPa, the cell viability is low. Note that the storage temperature is preferably −3 ° C. or higher in order to dispel the fear of freezing of water in the cells due to physical shock, and −1 ° C. or higher in terms of providing for experiments immediately after storage and cell viability. Is more preferable, and 1 degreeC or more is further more preferable. In addition, as described above, the optimum temperature varies depending on the cell type, but it is preferably 40 ° C. or lower, more preferably 37 ° C. or lower, in that it is used for experiments immediately after storage. The storage pressure is preferably 0.11 MPa or more in terms of cell viability, and more preferably 0.15 MPa or more in consideration of the risk of pressure leakage during storage. The maximum pressure is preferably 6.5 MPa or less, more preferably 5.5 MPa or less, from the viewpoint of maintaining the cell viability for a long period of time.

In addition, when storing many cells, it is preferable to pressurize the stored cells uniformly with hydrostatic pressure in order to achieve both the rapidity of the cell storage process and the high survival rate of the cells. In the method, cells to be stored are suspended in a liquid medium in a storage container having a material capable of transmitting external pressure to an internal substance and sealed, and the storage container is placed in a pressurized container with a liquid pressure medium. A method of applying pressure is preferred.

The storage container to which pressure is applied as described above or the pressurized container in which the storage container is stored is stored in a thermostatic chamber, a constant temperature room, or indoors if the storage temperature can be maintained.

The cell storage device of the present invention will be described more specifically as follows. First, the pressurized container has a valve for pressure adjustment, and the valve may serve both as pressurization and decompression, and the pressurization and decompression may be separate valves. The type of the valve is not particularly limited as long as it can withstand the pressure. For example, a gate valve, a ball valve, a needle valve, a stop valve, or the like can be used.

As a method for increasing the pressure inside the pressurized container to a desired pressure, there is a method using a compressor or the like as a fluid transfer device when the pressure medium is a gas, and a method using a liquid feed pump when the pressure medium is liquid. For the above reasons, it is preferable to pressurize with a liquid pressure medium using a liquid delivery device such as a liquid delivery pump. In the operation of returning the inside of the pressurized container to the atmospheric pressure in order to take out the preserved cells after storage, the pressure medium may be discharged by simply opening the valve, and the pressure may be reduced. The pressure medium may be discharged.

A preferred embodiment of the cell storage device of the present invention is configured as shown in FIG. 1 in order to uniformly apply pressure to the stored cells as described above. That is, a storage container made of a material capable of transmitting an external pressure to an internal substance, a pressurized container capable of storing one or a plurality of the storage containers, the liquid pressure medium, and the liquid pressure medium are stored. A storage tank, a liquid feed pump (fluid transfer device) for transferring the liquid pressure medium, and piping are basically constructed. In order to monitor the pressure application state, it is preferable that a pressure indicator such as a pressure gauge for displaying the internal pressure is attached to the pressurized container. Furthermore, in order to observe the inside of the pressurized container, a glass or transparent resin window may be appropriately attached.

The pressure adjusting valve is attached to the pressure vessel. After the pressure treatment, the valve is closed, and the piping and the liquid feed pump are removed to remove the pressure vessel (inside the cell Only storage containers can be transported. The pipe material is not particularly limited, and examples include stainless steel, steel, copper, brass, or vinyl chloride resin, or a flexible type such as a Teflon (registered trademark) tube, a silicon tube, or a pressure-resistant rubber tube. Tubes are also suitable.

The size of the cell storage device of the present invention is not particularly limited as long as it is theoretically possible in terms of production, but for the purpose of cell storage, a cell storage container capacity of 0.01 mL to several L is practical. Preferably 0.1 mL to 1 L. The pressurized container is designed in accordance with the size of the storage container and the number of the storage containers stored in the pressurized container.

According to the cell storage method and apparatus of the present invention described above, cells immediately after being collected from a living body or cells in culture can be quickly and easily placed in a storage environment. This is because, in the present invention, it is not necessary to set an extremely low temperature, it is not necessary to set an extremely high pressure, and the preparation of the storage solution and the mixing operation of the storage solution and cells are not required. Therefore, for later experiments or cell transplantation, if necessary, several types of cells or the same type of cells with different media can be quickly placed in a storage environment under the same conditions, improving the accuracy of comparative experiments. And is effective in maintaining biological functions.

According to the method and apparatus of the present invention, cells can be stored with a high survival rate for one day to several weeks, and even when releasing the storage state, the pressurized state is not extremely high pressure, so the atmospheric pressure can be reduced in a short time. Can be returned to. In addition, since the preserved cells are not frozen or the like, the preparation for the experiment can be quickly started by attaching the cells to a plate or the like immediately after the preservation period. The length of the storage period cannot be generally described because there is a difference in the strength of vitality or adaptability to environmental changes depending on the cell type, but it can be stored for 2 to 4 weeks.

As will be described in detail in the examples described later, by using the storage method of the present invention, the survival rate of the cells is dramatically increased as compared with the case of storing without applying pressure. The reason why this effect is obtained is not clear, but as it is known that tadpoles go into a nap state under high pressure, the cell is put into a nap state (anesthetic effect works) by pressurization, and the metabolism is slow. Therefore, it is presumed that the medium can be viable without changing the medium for a long time with little consumption of the medium.

Next, an embodiment of the present invention will be described below with reference to the drawings. FIG. 1 described above conceptually shows a typical example of the cell storage device of the present invention. FIG. 2 shows a state in which the storage container is housed in a pressurized container, and shows a cross-sectional view of both containers. In FIG. 2, the storage container is depicted as floating in the pressure medium. However, this is for ease of understanding, and is actually in contact with the inner wall of the pressurized container or applied. When the storage container support is provided in the pressure vessel, it is supported by the support. The storage tank 1 is filled with a liquid pressure medium and connected to the liquid feed pump 2 by a pipe 9. In this embodiment, water is mainly used as the liquid pressure medium, and a Teflon (registered trademark) tube is used as the pipe.

Cells, for example, rat neonatal brain primary culture system (2 × 10 ⁷ / dish) is cultured for 20-30 days, and after glial cells mature, trypsin (0.05%) is removed and stored in storage container 3 in liquid medium Suspend in and fill. As the liquid medium, various mediums for cell culture can be widely used. In this embodiment, a DMEM medium is used. Table 1 shows the composition of the DMEM medium manufactured by GIBCO used in this study. The storage container 3 has a cylindrical shape, the cylindrical portion is a silicone resin tube, and the upper and bottom surfaces of the cylinder are plugged into the silicone resin tube using a glass or Teflon (registered trademark) resin stopper. It is a structure. The top surface or bottom surface of the cylinder is plugged, and after filling with the cell suspension medium, the other surface is also plugged and sealed.

The storage container 3 enclosing the cells is placed in a pressurized container 4. In this embodiment, the pressurized container 4 has a cylindrical shape made of stainless steel, and is provided with an open / close hatch 7 on the upper surface or the bottom surface, and the storage container 3 is accommodated from the hatch. In the present embodiment, the storage container 3 is not particularly supported in the pressurized container 4, but a support body is provided in the pressurized container so that the storage container 3 can be supported at the center of the pressurized container, for example. It can also be done. In order to store a plurality of storage containers at the same time, it is also preferable to install the support in a plurality of pressurized containers.

In addition, although the thing with the viewport (observation window) 11 is shown as the opening-and-closing hatch in the pressurized container 4 of FIG. 1, the window may be installed in another position, The said window part Is not necessary. Furthermore, the open / close hatch is not essential, and the device structure may be any as long as the storage container 3 can be accommodated in the pressurized container 4.

The liquid feed pump 2 and the pressurized container 4 are connected by a Teflon (registered trademark) tube through a valve 5, and after storing the storage container 3 in the pressurized container 4 and closing the open / close hatch, the valve 6 is turned on. In an open state, the water in the storage tank 1 is fed into the pressurized container 4 by the liquid feed pump 2. Alternatively, water may be fed into the pressurized container before storing the storage container. When the inside of the pressurized container 4 is completely filled with water, the valve 6 is closed. In this state, while monitoring the pressure in the pressurized container 4 with the pressure indicator 8, further water is fed in until the desired pressure is reached. At this time, it is desirable that water as the pressure medium is maintained at a desired storage temperature.

The cell storage device may be installed in a temperature-controlled room in advance and stored at a desired temperature as it is after the operation is completed, or the pressurized container 4 containing the storage container 3 is removed from the liquid feeding pump or the like as described above. Only the pressurized container 4 (including the storage container 3) may be stored in a thermostat or the like.

In the present embodiment, the storage container 3 and the pressurized container 4 are both cylindrical. However, as described above, the storage container 3 may have a spherical shape or a bag shape such as an infusion bag. A rectangular parallelepiped or a cube having a hollow inside, a so-called cooler box-like shape may be used. That is, for example, a storage method may be used in which a plurality of drip bag-like storage containers are arranged in a cooler box-like pressurized container.

Since the storage device of the present invention can remove only the pressurized container 4 as described above, it can be easily transported even when the place where the cells are collected and the experiment place or the treatment place are separated. If necessary, the pressurized container can be further put into a thermostatic container such as a cooler box and transported while maintaining a desired temperature.

When the stored state is released and the cells are taken out, the valve 5 or 6 or both may be opened to discharge the liquid pressure medium (water in this embodiment) to return the pressure to atmospheric pressure. Alternatively, when it is desired to precisely control the pressure reduction speed when returning to the atmospheric pressure, the liquid pressure medium may be discharged at a desired speed using a liquid feed pump. If the temperature at the time of cell storage and the temperature at the time of treatment after storage are different, the temperature may be adjusted before returning the pressure to atmospheric pressure, or the temperature may be adjusted after returning the pressure to atmospheric pressure. Although it is good, it is preferable to return to atmospheric pressure after adjusting the temperature.

Hereinafter, the present invention will be described in more detail using examples. This experiment is roughly divided into three parts: separation of cultured cells, pressure treatment, and cell observation after storage.

<Experimental method and conditions>
1. As a cell of the isolated sample of the cultured cells, a rat neonatal brain primary culture system (2 × 10 ⁷ dish) was cultured for 20 to 30 days and the glial cells were matured. In addition, the conditions such as the number of cells are adjusted to be the same as possible by counting the cells, but the conditions are not necessarily the same depending on the lot of cells. Therefore, the same cells were divided into three each time, and for comparison, one was always stored under no pressure (ie, atmospheric pressure; about 0.1 MPa), and the other two were used as pressure storage samples.

(1) Primary culture system (2 × 10 ⁷ dish) glial cells removed from the rat neonatal brain were cultured in a 250 mL culture flask for 20-30 days using a CO ₂ incubator (WAKENYAKU Model 9100, CO ₂ concentration 5%) at 37 ° C. And matured using DMEM medium. The glial cells adhere to the bottom of the flask and survive with the protrusions extended.

(2) In order to take out the cells from the culture flask of (1), first, the medium was sucked out of the flask with an aspirator.

(3) To further wash away the medium adhering to the cells, 6 mL of PBS (phosphate buffer solution containing physiological saline) was added to the flask and shaken gently, and then the washing solution was removed by sucking with an aspirator.

(4) 2 mL of 0.05% trypsin was added to the flask in order to detach cells adhering to the flask using an enzyme reaction.

(5) To make trypsin work efficiently, it was kept in a constant temperature bath at 37 ° C. for 2 to 3 minutes. Thereafter, the cells were removed from the incubator, and the cells were detached by tapping the flask.

(6) 2 mL of horse serum and 6 mL of PBS were added to the flask to stop trypsin activity.

(7) Transfer the solution from the flask to a centrifuge tube, and centrifuge at 800 rpm for 7 minutes using a Hitachi centrifuge, himac-CT4i to precipitate the detached cells, taking care not to absorb the cells that have settled on the bottom. The supernatant was sucked up with an aspirator.

(8) 1.5 mL of DMEM medium was added to the centrifuge tube to sufficiently suspend the cells.

(9) In the subsequent experiments, the cell suspension solution is used by dividing it into several pieces. At this time, in order to make the concentration of the cell solution (number of cells) the same in each subdivision, the cell suspension solution is used as much as possible. It is necessary that the cells are individually and uniformly dispersed in the medium. Therefore, in order to remove most of the aggregated cells, it was filtered with a 100 μm filter, and the filtrate (cell suspension) in which the cells were dispersed in a single unit was collected. .

(10) A small amount of the cell suspension was taken with a pipetman, placed in a hemocytometer so as to be uniformly distributed, and the number of cells in the field of view was counted with a microscope according to a conventional method, and the number of cells was calculated.

(11) Based on the number of cells calculated in (10) above, the concentration was adjusted with DMEM medium so that the cell concentration was 4 × 10 ⁶ to 6 × 10 ⁶ cells / 300 μL.

(12) 300 μL of the cell suspension of (11) was weighed out uniformly, put into a silicon tube as a storage container, and sealed with a Teflon (registered trademark) lid, taking care not to enter air.

2. Pressure treatment (storage conditions)
(13) Water (pressure medium) set to each storage temperature was put into a pressurized container (maximum pressurizable pressure 15 MPa). Said temperatures are either 0 ° C., 4 ° C., 20 ° C. or 37 ° C., respectively. When the storage temperature was 0 ° C., a solution in which a small amount of ethylene glycol was added to water was used in order to prevent the pressure medium from icing (hereinafter, this example and the mixed solution including the water-ethylene glycol mixture were also compared). An example pressure medium is generically referred to as water).

(14) The storage container prepared in (12) was placed in the pressurized container filled with water.

(15) Using a Shimadzu HPLC liquid feed pump LC6A, water was fed and the inside of the pressurized container was pressurized with hydrostatic pressure. Pressurization was carried out to a desired storage pressure at a pressurization rate of 0.15 to 5.0 MPa / min. The pressure was measured with a pressure gauge manufactured by Yamazaki Seisakusho. The storage pressure was the pressure shown in Tables 2 to 4 below.

(16) The sample was stored for 1 day, 4 days or 5 days under each set storage temperature and set storage pressure.

3. Cell observation after storage (17) After removing the storage container from the pressurized container, remove the lid of Teflon (Registered Trademark), suspend the cells uniformly, and suck up with a liquid medium with a pipette, and a 15 mL Corning tube Moved to.

(18) Add 3 mL of serum-containing DMEM medium to the above-mentioned Corning tube to dilute the cell concentration to 1.5 × 10 ⁵ to 2 × 10 ⁵ cells / 300 μL, and then add it to 3 holes in the 12-well culture plate. Separately seeded with the medium.

(19) The culture plate was placed in a 37 ° C. CO ₂ incubator, and the preserved cells were cultured for 2 days.

(20) The cell viability was evaluated as follows. Since the cells used in this example have a property of forming a spherical shape when detached from the culture plate, the cells are spherical and suspended in a liquid medium during storage. If the cells are alive after storage, they are observed to adhere to the bottom of the plate in about 1 to 2 days and grow with protrusions when they are seeded again on the culture plate. On the other hand, if it is not alive, it is spherical and remains suspended in the liquid medium. Therefore, the number of cells adhering to the bottom of the plate after the culture of (19) is counted with a microscope to obtain the number of viable cells. On the other hand, without storing the cell suspension prepared in “1. Cultured cell separation”, the number of viable cells in the case where the treatments (18) and (19) were performed was set as a blank, and the number of viable cells corresponding thereto was determined. The cell survival rate was expressed as a percentage.

The quality of cell viability was evaluated according to the following criteria. That is, ◎ (survival rate: 60% or more), ○ (survival rate: less than 60% and 30% or more), △ (survival rate: less than 30% and 10% or more), × (survival rate: less than 10%) did.

Examples 1 to 4
According to the above experimental method and conditions, storage at 4 ° C., storage pressure of 0.11, 1.0, 7.5, or 10.0 MPa (in the order of Examples 1, 2, 3, 4, and so on) for 1 day. After that, the cell viability was measured. As a result, in all cases, the cell viability was good at 60% or more.

Comparative Examples 1 and 2
The sample was stored under the same conditions as in Example 1 except that the storage pressure was 0.1 or 14 MPa (Comparative Examples 1, 2, and so on). As a result, the cell survival rate was about 20% under 0.1 MPa and less than 10% at 14 MPa.

Example 5
It was stored under the same conditions as in Example 1 except that the storage temperature was 37 ° C. and the storage pressure was 10 MPa. As a result, the cell viability was about 50%.

Comparative Examples 3-4
It was stored under the same conditions as in Example 1 except that the storage temperature was 41 ° C. and the storage pressure was 0.11 or 10 MPa. As a result, the cell viability in each case was almost 0%.

Examples 6-7
According to the above experimental method and conditions, the cell viability was measured after storing for 4 days at a storage pressure of 0.11 or 10.0 MPa at a storage temperature of 0 ° C. As a result, in all cases, the cell viability was good at 60% or more.

Comparative Examples 5-6
It was stored under the same conditions as in Example 6 except that the storage pressure was 0.1 or 11 MPa. As a result, the cell viability in each case was less than 10%.

Examples 8-11
It was stored under the same conditions as in Example 6 except that the storage temperature was 4 ° C. and the storage pressure was 0.2, 0.5, 1.0, or 6.5 MPa. As a result, the cell viability when the storage pressure was 0.2, 0.5, or 1.0 MPa was good at 60% or more. In the case of 6.5 MPa, it was about 50%.

Comparative Example 7
It was stored under the same conditions as in Example 6 except that the storage temperature was 4 ° C. and the storage pressure was 0.1 MPa. As a result, the cell survival rate was almost 0%.

Example 12
It was stored under the same conditions as in Example 6 except that the storage temperature was 20 ° C. and the storage pressure was 0.6 MPa. As a result, the cell survival rate was good at 60% or more.

Comparative Example 8
It was stored under the same conditions as in Example 6 except that the storage temperature was 20 ° C. and the storage pressure was 11 MPa. As a result, the cell survival rate was almost 0%.

Example 13
It was stored under the same conditions as in Example 6 except that the storage temperature was 37 ° C. and the storage pressure was 0.11 MPa. As a result, the cell survival rate was good at 60% or more.

Example 14
According to the experimental method and conditions described above, the cell viability was measured after storage for 5 days at a storage temperature of 4 MPa at a storage temperature of 3.0 MPa. As a result, the cell viability was about 50%.

Comparative Examples 9-10
It was stored under the same conditions as in Example 14 except that the storage pressure was 0.1 or 11 MPa. As a result, the cell viability in each case was almost 0%.

Table 2 shows the results for a storage period of 1 day.

Table 3 shows the results for a storage period of 4 days.

Table 4 shows the results for a storage period of 5 days.

Cells are subjected to experiments for various purposes in a wide range such as medical fields, pharmaceutical fields, livestock fields, and academic fields such as biochemistry. It also plays an important role in transplantation treatment. In an experiment using these cells, for example, a culture experiment, the experiment may be temporarily interrupted to be stored. In such cases, simply refrigeration not only lowers cell viability, but some cells cannot be preserved. On the other hand, when the cells are frozen or a storage solution is used, as described above, there are cases where the method cannot be employed due to lack of rapid storage and resumption of experiments. According to the storage method of the present invention, these disadvantages can be solved, and storage and experiments can be resumed quickly and easily. For example, primary cultured cells that maintain their properties in vivo are desired to be used for experiments quickly. It is possible to meet the demand for cell preservation in a wide range of fields.

DESCRIPTION OF SYMBOLS 1 ... Liquid pressure medium storage tank, 2 ... Liquid feed pump (fluid transfer apparatus), 3 ... Storage container, 4 ... Pressurization container, 5, 6 ... Valve, 7 ... Opening-closing hatch, 8 ... Pressure indicator, 9 ... Piping 10 ... Cells suspended in liquid medium, 11 ... Observation window

Claims

Under the storage conditions, the storage temperature is higher than the crystallization temperature of intracellular water to be stored, and is not more than the optimum temperature of cell activity, and the storage pressure is 0.11 to 10 MPa.
Cell preservation method.
The cells are seeded or suspended in a medium that is liquid under the storage conditions.
The cell storage method according to claim 1.
Enclosing the cells in a storage container comprising a material capable of transmitting external pressure to the internal substance, and pressurizing the cells through the storage container with a pressure medium;
The cell preservation method according to claim 1 or 2.
Encapsulating cells in the storage container;
Storing one or more of the storage containers enclosing the cells in a pressurized container;
Maintaining the inside of the pressurized container at the temperature;
And maintaining the pressure by filling the pressurized container with a pressure medium, and pressurizing the cells in the storage container,
The cell storage method according to claim 3.
The pressure medium is a liquid;
The cell storage method according to claim 3.
The pressure medium is a liquid;
The cell preservation method according to claim 4.
The storage temperature is 1 ° C. to 37 ° C., and the storage pressure is 0.11 to 6.5 MPa.
The cell storage method according to claim 1.
A pressurized container capable of maintaining pressure by filling with a pressure medium;
Storage of one or more cells that can be stored in the pressurized container and can pressurize cells enclosed inside by providing a material capable of transmitting external pressure to the internal substance A container,
Cell storage device.
Adjusting the pressure in the pressurized container by feeding the pressure medium into the pressurized container by a fluid transfer device;
The cell storage device according to claim 8.
The pressure medium is a liquid;
The cell storage device according to claim 8 or 9.
By controlling the feeding and discharging of the pressure medium by the fluid transfer device, the pressure increase / decrease speed can be adjusted,
The cell storage device according to claim 9.
By controlling the feeding and discharging of the pressure medium by the fluid transfer device, the pressure increase / decrease speed can be adjusted,
The cell storage device according to claim 10.