JP7265268B2 - Biological sample storage container - Google Patents

Description

Article 30, Paragraph 2 of the Patent Law applies Publisher name: The Japanese Society for Regenerative Medicine Publication name: The 16th General Assembly of the Japanese Society for Regenerative Medicine Program/Abstract Page 405 Date of publication: February 1, 2017 March 7-9, 2017 Presented at the 16th Annual Meeting of the Japanese Society for Regenerative Medicine

TECHNICAL FIELD The present invention relates to a container for storing a biological sample such as a cell sheet, and a method for freezing and thawing a biological sample using the container.

Cell sheets are being widely applied to regenerative medicine, but the time lag required for transplantation is unavoidable due to the time required for culturing. Therefore, a vitrification and cryopreservation method has been developed as a technique for long-term storage of cell sheets in a state where they can be used immediately when needed (Non-Patent Document 1).

In the preservation method described in Non-Patent Document 1, the cell sheet is immersed in an equilibrium solution, then in a vitrification solution, and then exposed to liquid nitrogen vapor to freeze it. In addition, the frozen cell sheet is thawed by heating the cell sheet on an electric heating plate and then immersing it in a thawing solution, a dilution solution, and a washing solution in that order.

M. Maehara et al., BMC Biotechnology 13(58), 2013

The preservation method described in Non-Patent Document 1 is an excellent technique that enables cryopreservation of cell sheets while maintaining the structure of the cell sheet and the high viability of the constituent cells. However, there are some problems. For example, in this method, the immersion operation in each liquid is performed by moving the cell sheet from a culture dish containing a certain immersion liquid to a culture dish containing another immersion liquid using tweezers or the like. For this reason, there is a risk that various germs will be mixed in during the movement. Moreover, there is a possibility that the cell sheet may be damaged by pinching the cell sheet with tweezers. Furthermore, skill is required for operation, and a new method that does not depend on the skill level of the operator is required. In order to solve these problems, and to enable cryopreservation of a large amount of cell sheets, the inventors have come to the conclusion that it is necessary to consider automating the cryopreservation method.

An object of the present invention is to solve such problems of conventional cell sheet vitrification cryopreservation methods.

As a result of intensive studies to solve the above problems, the present inventors have found that a cell sheet is housed in a sealable bag, two tubes having small holes are placed at the periphery of the bag, and one of the tubes By injecting the immersion liquid into the bag through the pipe and discharging the immersion liquid through the other pipe, it was found that all of the hygiene problems, breakage problems, and automation problems in the immersion operation described above can be solved. Completed.

That is, the present invention provides the following [1] to [6].
[1] A biological sample storage container having a container body for containing a biological sample, an injection member for injecting a liquid from the outside of the container into the inside of the container, and a discharge member for discharging the liquid from the inside of the container to the outside of the container, The member and the discharge member are placed at a position such that a liquid flow occurs between the injection member and the discharge member, and the liquid flow contacts the biological sample contained in the container body. A biological sample storage container characterized by comprising:

[2] In [1], the injection member and the discharge member are pipes, a portion of which is positioned inside the container body, and the portion is provided with a plurality of small holes. A biological sample storage container as described.

[3] The biological sample storage container according to [1] or [2], characterized in that the injection member and the discharge member are installed at the periphery of the container body.

[4] The biological sample storage container according to any one of [1] to [3], wherein the biological sample is a cell sheet.

[5] A biological sample freezing method comprising the following steps (1) to (4):
(1) A step of storing a biological sample in the container body of the biological sample storage container according to any one of [1] to [4];
(2) a step of injecting the equilibrium liquid from the injection member into the container body, bringing it into contact with the biological sample, and then discharging it from the discharge member;
(3) a step of injecting the vitrification liquid from the injection member into the container body, bringing it into contact with the biological sample, and then discharging the liquid inside the container from the discharge member;
(4) A step of cooling the biological sample storage container and freezing the biological sample.

[6] A method of thawing a frozen biological sample, comprising the following steps (1) to (4):
(1) Step of heating the biological sample storage container according to any one of [1] to [4], in which a frozen biological sample is stored in the container body; After injecting into and contacting with the biological sample, discharging the liquid inside the container from the discharging member;
(3) a step of injecting the diluent into the container body through the injection member, bringing it into contact with the biological sample, and then discharging the liquid inside the container from the discharge member;
(4) A step of injecting the cleaning liquid from the injection member into the container main body, bringing it into contact with the biological sample, and then discharging the liquid inside the container from the discharge member.

The present invention provides a container for storing a biological sample such as a cell sheet, and a method for freezing and thawing a biological sample using the container.

It is a figure which shows an example of the biological sample storage container of this invention. FIG. 4 is a diagram showing another example of the biological sample storage container of the present invention; It is a figure which shows the biological sample storage container used in the Example. Fig. 4 is a morphological photograph of a cell sheet (A) after vitrification freezing and thawing and a cell sheet (B) without vitrification freezing.

The present invention will be described in detail below.
[1] Biological sample storage container The biological sample storage container of the present invention comprises a container body for containing a biological sample, an injection member for injecting liquid from the outside of the container into the inside of the container, and a discharge member for discharging the liquid from the inside of the container to the outside of the container. wherein the injection member and the discharge member are positioned such that a liquid flow occurs between the injection member and the discharge member, and the liquid flow is contained in the container body. It is characterized in that it is installed at a position such that it comes into contact with the

The container body may be of any type as long as it can contain the biological sample, but a bag-like container is preferred. The bag-shaped container is preferably provided with an opening for taking in and out the biological sample, and the opening is preferably openable and closable with a zipper or the like. As this bag-like container, a commercially available storage bag with a zipper can be used. The container body is not limited to a bag-like container, and may be, for example, a container in which films are adhered to the upper and lower surfaces of a picture-frame-like frame. The shape of the container body may be any shape, but a rectangular shape is preferable in terms of ease of installation of the injection member and the discharge member. Any material may be used for the container body, but a material that is transparent and has good thermal conductivity is preferable. When the container body is a bag-like container, the material of the container body is, for example, low-density polyethylene, medium-density polyethylene, ultra-high molecular weight polyethylene, polyethylene-polytetrafluoroethylene copolymer, polyethylene-1,2- A dichloroethane copolymer and the like are preferred. The size of the container body can be determined according to the biological sample to be accommodated. When the biological sample is a cell sheet and the container body is a rectangular bag-like container, the rectangle preferably has a long side of 30 to 350 mm and a short side of 10 to 100 mm.

The injection member may be of any type as long as it can inject liquid from the outside of the container into the inside of the container. is preferably a tube provided with a plurality of small holes. In such a tube, the liquid can be injected from the outside of the container into the inside of the container by allowing the liquid to flow in from the outside of the container body and to flow out from the small hole. The diameter of the tube is not particularly limited as long as the liquid can be injected from the outside of the container into the inside of the container, but it is preferably 0.5 to 5 mm. The material of the tube is not particularly limited as long as the liquid can be injected from the outside of the container into the inside of the container, but Teflon (registered trademark), polyethylene, silicon, and the like are preferable. The positions of the small holes provided in the tube are not particularly limited, but the small holes are preferably provided at regular intervals throughout the portion located inside the container body. In this case, the interval between the small holes is not particularly limited, but it is preferably 5 to 15 mm. The inner diameter of the small hole provided in the tube is not particularly limited, but it is preferably 0.5 to 3 mm. The shape of the tube is not particularly limited, and may be a linear shape, a polygonal shape, or a curved shape. The tube may be branched or integral with the tube used as the evacuation member. One of the ends of the tube opens outside the container body and the other one usually opens inside the container body. However, both ends may open to the outside of the container body. When the tube is branched, it has three or more ends, and all of them may be open to the outside of the container body, or only some of them may be open to the outside of the container body. Further, the bore diameter of the tube or the inner diameter of the small holes may not be uniform, and may be formed so that the amount of liquid flowing out from each small hole is substantially uniform.

The discharge member may be of any type as long as it can discharge the liquid from the inside of the container to the outside of the container. is preferably a tube provided with a plurality of small holes. In such a tube, the liquid can be discharged from the inside of the container to the outside of the container by allowing the liquid to flow into the small hole and to flow out of the container body. The diameter of the tube, the material of the tube, the shape of the tube, the position of the ostium, and the inner diameter of the ostium may be similar to the tube used as the injection member.

From the viewpoint of manufacturing efficiency, the injection member and the discharge member may be completely the same, but they may be partially different in shape and material.

The injection member and the discharge member are installed at a position such that a liquid flow occurs between the injection member and the discharge member and the liquid flow contacts the biological sample contained in the container body. It is Here, "a liquid flow occurs between the injection member and the discharge member" means that the liquid moves between the injection member and the discharge member without staying there. The flow of the liquid may be substantially linear, or may be curved within a range in which the liquid does not stagnate.

By arranging the injection member and the discharge member at positions where liquid flows between them, it is possible to quickly replace the liquid in the container body with the liquid to be newly injected. become. On the other hand, when the injection member and the discharge member are positioned so that no liquid flow occurs between them, for example, a cell culture vessel (for example, the cell culture vessel described in Japanese Patent Application Laid-Open No. 2008-22715) ), etc., where an inlet (injection member) and an outlet (discharge member) are adjacent to one side of a rectangular container, the new liquid injected from the inlet is originally inside the container. Since it will be mixed with a certain liquid, the liquid that is discharged from the outlet is not the liquid that was originally in the container, but the liquid that is a mixture of the new liquid and the liquid that was originally in the container, except immediately after the start of injection. . Therefore, it takes a very long time to replace the liquid.

The storage container of the present invention is mainly used in the vitrification freezing method, and in the vitrification freezing method, the rapid exchange of liquid as described above is very important. This is because, in the vitrification freezing method, a biological sample is sequentially immersed in a plurality of different liquids (e.g., equilibrium solution, vitrification solution, etc.). This is because the substrate is immersed in a mixture of both liquids for a long period of time instead of being immersed in the vitrification liquid. On the other hand, when replacing the liquid medium in the cell culture vessel, it is not necessary to quickly replace the new liquid medium with the old liquid medium, and the new liquid medium may harm the cells. Rather, it is preferable to replace the liquid over a certain amount of time. In studying the storage container of the present invention with the above findings as one of the purposes of automating vitrification cryopreservation of biological samples, the inventors found that there is no liquid flow between the injection member and the discharge member. It has now been found that it is preferable to position both members so that they occur. Therefore, it is preferable to install the injection member and the discharge member so that a liquid flow occurs between them, even when the storage container of the present invention is used in the vitrification method.

This occurs because the liquid surrounding the biological sample may not be replaced with fresh liquid if the biological sample does not come into contact with the liquid flow that occurs between the injection member and the ejection member. To prevent this, the injection member and the ejection member are positioned so that the flow generated between the two members contacts the biological sample contained in the container body.

When the injection member and the discharge member are the pipes described above, they are usually placed on the periphery of the container body to allow a liquid flow to occur between the two members. comes into contact with the biological sample, and is preferably placed in such a position. Specifically, when the container body is a rectangular bag-shaped container, it is preferable to install the injection member and the discharge member along opposite sides of the rectangle.

The container of the present invention is a container for preserving a biological sample, preferably a container for cryopreserving a biological sample, and more preferably a container for vitrifying and cryopreserving a biological sample. Here, the term "biological sample" refers to a biological sample, such as cells, spheroids, tissues, organs, fertilized eggs, embryos, or parts thereof (eg, sections). A biological sample may be of human origin or non-human animal origin. Animals other than humans include, for example, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, and monkeys.

Although the biological sample to be preserved is not particularly limited, it is preferably a cell sheet. As used herein, the term “cell sheet” refers to an aggregate of cells formed into a sheet by adhering or aggregating cells. Cell sheets of various cells such as chondrocytes, corneal epithelial cells, skin cells, oral mucosal epithelial cells, bladder epithelial cells, cardiomyocytes, and periodontal ligament cells have been produced so far. A cell sheet of any cell can be stored.

The biological sample may be stored in the container body as it is, or may be stored together with an appropriate protective material, scaffolding material, or the like. Specifically, the biological sample can be embedded in a gel, encapsulated, fixed on a chip, or coated with a permeable membrane, and then housed in the container body.

Recently, a method has been reported for artificially constructing microscale fiber-shaped cell tissue by culturing a mixed solution of cells and collagen while flowing them through microtubules (H. Onoe, et al. , Nature Materials, Vol. 12, pp. 584-590, 2013). Such fiber-shaped cell tissue can also be stored in the container of the present invention.

The present inventors have reported a method of vitrifying and freezing mouse or pig embryos in hollow fibers (H. Matsunari, et al., Journal of Reproduction and Development Vol. 58 (2012) No. 5 p. 599-608). Hollow fibers containing such biological samples can also be stored in the container of the present invention. In the above method, about 20 to 40 biological samples (embryos) can be placed in one hollow fiber. It is possible to store a very large number of biological samples. When storing such hollow fibers, the hollow fibers are preferably arranged in the container body so as to be parallel to the liquid flow. By arranging in this way, it is possible to prevent the hollow fibers from interfering with the flow of the liquid.

By embedding the biological samples to be preserved in one plate-like gel together and then housing it in the container main body, it is possible to collectively cryopreserve many biological samples. Moreover, when the gel is a gel such as gelatin that melts with heat, the biological sample can be easily taken out from the gel by heating. Such plate-shaped gels can also be stored in the container of the present invention. Since the biological samples to be preserved are to be embedded together in this plate-shaped gel, the size of the gel is almost the same as that of the main body of the container. Specifically, for example, when the plate-shaped gel is substantially rectangular, the thickness is 0.5 to 5 mm, the long side is 10 to 200 mm, and the short side is 5 to 150 mm.

The liquid to be injected into the injection member is not particularly limited, but since the container of the present invention is mainly used for vitrification cryopreservation, the liquid is usually a liquid used for vitrification cryopreservation, such as equilibrium liquid, vitrification liquid, These include melting liquids, diluents, washing liquids, and the like.

Next, an example of the biological sample storage container of the present invention will be described with reference to FIG. This biological sample storage container consists of a rectangular bag 1 , an injection tube 3 and an ejection tube 4 . An opening 2 that can be opened and closed by a zipper or the like is provided on one short side of the bag 1 , and a biological sample is put into the bag 1 through this opening 2 . The injection pipe 3 and the discharge pipe 4 are fixed inside the bag 1 along the long sides of the bag 1 . One end of the injection tube 3 is inside the bag 1 while the other end is outside the bag 1 . An injection port 5 is provided at this external end. A plurality of small holes 7 are provided at regular intervals in a portion of the injection tube 3 located inside the bag 1 . The discharge tube 4, like the injection tube 3, has one end inside the bag 1 and the other end outside the bag 1. FIG. This external end is provided with a discharge port 6 . Although not shown in this figure, a portion of the discharge tube 4 located inside the bag 1 is also provided with a plurality of small holes 7 at regular intervals, similar to the injection tube 3 .

When the biological sample is placed in the biological sample storage container and vitrified and frozen, the equilibrium solution and the vitrification solution are sequentially injected from the inlet 5 . The equilibrium solution injected from the injection port 5 passes through the injection tube 3, flows out from the small hole 7, and moves inside the bag 1. As shown in FIG. The equilibrium liquid flowing out from the small hole 7 of the injection tube 3 forms a flow (arrow in the figure) without staying and flows into the small hole 7 of the discharge tube 4 . Although not shown in this figure, the bag 1 contains a biological sample, and the equilibrium liquid flowing out from the small hole 7 of the injection tube 3 contacts the biological sample and then flows into the small hole of the discharge tube 4 . It flows into hole 7 . As a result, the biological sample is immersed in the equilibrium solution necessary for vitrification freezing. The equilibrium liquid that has flowed into the small hole 7 of the discharge pipe 4 passes through the discharge pipe 4 and is discharged from the bag 1 through the discharge port 6 . After the equilibration liquid is injected, the vitrification liquid is injected from the injection port 5 . The injected vitrification liquid is discharged from the outlet 6 to the outside of the bag 1 in the same manner as the equilibrium liquid. Thereby, immersion of the biological sample in the vitrification liquid required for vitrification freezing is performed. At this time, a flow of vitrifying liquid occurs between the small holes 7 of the injection tube 3 and the small holes 7 of the discharge tube 4, the small holes 7 being provided throughout the portion of these tubes located inside the bag 1. The flow of vitrifying liquid occurs throughout the interior of the bag 1 because it is closed. For this reason, the equilibrium liquid existing inside the bag 1 is quickly discharged and replaced with the vitrification liquid, so that the time during which the biological sample is immersed in the mixture of the equilibrium liquid and the vitrification liquid is minimized. suppressed. Therefore, by sequentially injecting the above-described equilibrium solution and vitrification solution, the biological sample is transferred from the container containing the equilibrium solution to the container containing the vitrification solution, which is substantially the same as the operation performed in the conventional vitrification freezing. The same operation was performed on

In the injection and discharge of the equilibrium liquid, the vitrification liquid, the melt liquid, the dilution liquid, the washing liquid, and the like, the same kind of liquid may be injected and discharged multiple times as necessary.

Another example of the biological sample storage container of the present invention will be described with reference to FIG. Like the container shown in FIG. 1, this biological sample storage container also comprises a rectangular bag 1, an injection tube 3, and a discharge tube 4, but the injection tube 3 and discharge tube 4 are integrated. As with the container shown in FIG. 1, one short side of the bag 1 is provided with an opening 2 that can be opened and closed by a zipper or the like. The injection tube 3 consists of a portion fixed along one long side of the bag 1 and a portion fixed along two short sides, and is fixed along the two short sides. The part that is fixed branches off from the fixed part along one of the long sides near two corners where the long side and the short side intersect. Two ends of the part fixed along the long side of the injection tube 3 are outside the bag 1, and an injection port 5 is provided at these ends. A discharge tube 4 is fixed along the other long side of the bag 1 and its two ends are outside the bag 1 . A discharge port 6 is provided at these ends. The discharge pipe 4 is connected to the injection pipe 3 near two corners where the long side and the short side meet, whereby the discharge pipe 4 is integrated with the injection pipe 3 . A plurality of small holes 7 are provided at equal intervals in the portions of the injection tube 3 and the discharge tube 4 located inside the bag 1 (the small holes provided in a part of the injection tube 3 and the discharge tube 4 are not shown in the figure). In FIG. 2, it is preferable that the injection pipe 3 and the discharge pipe 4 are not connected inside the pipes so that the liquids in both pipes do not mix.

By sequentially injecting the equilibrium liquid and the vitrification liquid into this biological sample storage container, the biological sample can be sequentially immersed in the equilibrium liquid and the vitrification liquid in the same manner as the container shown in FIG. However, in this container, in addition to the flow of liquid from the part fixed along the long side of the injection tube 3 to the discharge tube 4, the liquid flows from the part fixed along the short side of the injection tube 3 Since the liquid also flows into the discharge tube 4, when the liquid to be injected is switched from the equilibrium liquid to the vitrification liquid, the equilibrium liquid existing inside the bag 1 can be discharged more quickly and replaced with the vitrification liquid. can be done.

[2] Freezing method The biological sample freezing method of the present invention is characterized by including steps (1) to (4) described below. Step (1), step (2), step (3), and step (4) are performed in this order. Step (2) and step (3) may be performed multiple times.

In step (1), a biological sample is accommodated in the container body of the biological sample storage container.

The accommodation of the biological sample can be performed according to the container. For example, if the container is provided with an opening that can be opened and closed with a zipper or the like, the biological sample can be accommodated through the opening.

In step (2), the equilibrium liquid is injected from the injection member into the container body, brought into contact with the biological sample, and then discharged from the discharge member.

The equilibration solution used may be similar to that used in known vitrification freezing methods (eg, M. Maehara et al., BMC Biotechnology 13(58), 2013). For example, a medium in which the biological sample was cultured, to which ethylene glycol and dimethylsulfoxide are added, can be used as the equilibrium solution. The concentration of ethylene glycol contained in the equilibrium solution can be 1-20% by weight, preferably 5-15% by weight. The concentration of dimethylsulfoxide contained in the equilibrium solution can be 1-20% by weight, preferably 5-15% by weight. A medium to which ethylene glycol or dimethylsulfoxide is added can be appropriately selected according to the biological sample. Specific examples of media include DMEM medium, RPMI1640 medium, and TCM199 medium.

The temperature of the equilibrium solution may be the same as that used in known vitrification freezing methods, and may be, for example, 0 to 30°C.

The time for which the biological sample is brought into contact with the equilibrium solution may be the same time as the immersion time in the equilibrium solution in the known vitrification freezing method, and may be, for example, 1 to 30 minutes.

In step (3), the vitrification liquid is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid in the container is discharged from the discharge member.

The vitrification liquid used may be the same as that used in known vitrification freezing methods. For example, ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose are added to the medium in which the biological sample was cultured, and this can be used as the vitrification fluid. The concentration of ethylene glycol contained in the vitrification liquid can be 10-25% by weight, preferably 15-20% by weight. The concentration of dimethylsulfoxide contained in the vitrification liquid can be 10-25% by weight, preferably 10-20% by weight. The concentration of carboxylated polylysine contained in the vitrification liquid can be 1-15% by weight, preferably 5-10% by weight. The concentration of sucrose contained in the vitrification solution can be 0.1-2M, preferably 0.5-1M.

The temperature of the vitrification liquid may be the same as that used in known vitrification freezing methods, and may be, for example, 0 to 37°C.

The time for which the vitrification liquid is brought into contact with the biological sample may be the same as the immersion time in the vitrification liquid in the known vitrification freezing method, and can be, for example, 1 to 30 minutes.

In step (4), the biological sample storage container is cooled to freeze the biological sample.

The method for freezing the biological sample may be any method used in the known vitrification freezing method. Freezing the biological sample in the nitrogen gas phase is preferred.

[3] Thawing Method The method of the present invention for thawing a frozen biological sample is characterized by including steps (1) to (4) described below. Step (1), step (2), step (3), and step (4) are performed in this order. Step (2), step (3), and step (4) may be performed multiple times.

In step (1), the biological sample storage container, in which the frozen biological sample is stored in the container body, is heated.

The temperature and heating time during heating are the same as those used in known thawing methods after vitrification freezing (e.g., M. Maehara et al., BMC Biotechnology 13(58), 2013). Well, for example, it can be 20-45° C. for 1-60 seconds.

In step (2), the melted liquid is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid in the container is discharged from the discharge member.

The thawing solution used may be the same as that used in known thawing methods after vitrification freezing. For example, a medium in which the biological sample was cultured and sucrose added can be used as the thawing solution. The concentration of sucrose contained in the melt can be 0.1-2M, preferably 0.3-1M.

The temperature of the thawing solution may be the same as that used in known thawing methods after vitrification freezing, and may be, for example, 25 to 37°C.

The time for which the biological sample is brought into contact with the thawing solution may be the same time as the immersion time in the thawing solution in the known thawing method after vitrification freezing, and may be, for example, 0.5 to 3 minutes.

In step (3), the diluent is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid in the container is discharged from the discharge member.

The diluent used may be the same as that used in known thawing methods after vitrification freezing. For example, a medium in which the biological sample was cultured and sucrose added can be used as the diluent. The concentration of sucrose contained in the diluent can be 0.1-2M, preferably 0.3-1M.

The temperature of the diluent may be the same as that used in known thawing methods after vitrification freezing, and may be, for example, 20 to 37°C.

The time for which the biological sample is brought into contact with the diluent may be the same as the immersion time in the diluent in the known thawing method after vitrification freezing, and can be, for example, 1 to 10 minutes.

In step (4), the washing liquid is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid in the container is discharged from the discharge member.

The washing liquid used may be the same as that used in known thawing methods after vitrification freezing. For example, the medium in which the biological sample was cultured can be used as the washing liquid.

The temperature of the washing solution may be the same as that used in known thawing methods after vitrification freezing, and may be, for example, 20 to 37°C.

The time for which the biological sample is brought into contact with the washing liquid may be the same as the immersion time in the diluent in the known thawing method after vitrification freezing, and may be, for example, 1 to 10 minutes.

[4] Method of Freezing and Thawing Plate-shaped Gel A method of embedding a biological sample such as cells or spheroids in a gel and vitrification freezing has been known for some time. However, the gel to be frozen in such a method is very small, and a method for vitrifying and freezing a large gel such as the plate-shaped gel described above has not been known. Therefore, the method of vitrifying and freezing such a plate-shaped gel without storing it in the biological sample storage container and the method of melting it are novel methods, and the present invention provides such a method. Also includes Specifically, the plate-shaped gel is immersed in an equilibrium solution and a vitrification solution in order, and then vitrified and frozen, and the plate-shaped gel that has been vitrified and frozen is heated, melted, diluted, And the method of sequentially immersing in the cleaning solution is also included in the present invention.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

〔experimental method〕
(1) Preparation of cell sheet Japanese white rabbit knee cartilage-derived primary cultured cells (Cosmo Bio) were seeded in a temperature-responsive culture dish (Upcell, CellSeed), and the growth medium (20% FBS-containing DMEM/F12 medium) at 37°C and 5% _CO2 . When the cells reached confluence, the growth medium was replaced with a differentiation medium (RMI1640 medium containing 10% FBS and 100 μM L-ascorbic acid) and culture continued.

Two weeks after seeding, monolayer cell sheets were formed, which were stacked to form two layers and cultured for another week.

(2) Fabrication of cell sheet storage bag Put two tubes (made of Teflon, inner diameter 1.7 mm) in a polyethylene zipper bag (110 x 85 mm, film thickness 0.063-0.064 mm), one end outside the bag. I inserted it so that it sticks out. The inserted tube was fixed by heat sealing along the long side of the bag. Small holes with an inner diameter of 0.6 to 0.8 mm were provided at intervals of 5 to 10 mm in the portion of the tube inserted into the bag. FIG. 3 shows the appearance of this cell sheet storage bag.

(3) Freezing and thawing of the cell sheet The zipper of the storage bag was opened, and the cell sheet was put inside the storage bag. The cell sheet was sandwiched between two nylon meshes (50×50 mm, thread diameter 95 μm, mesh size 140 μm) and housed while being protected by these.

Place the storage bag on a shaker, and equilibrate solutions (10% ethylene glycol, 10% DMSO, and 20% bovine TCM199 solution containing serum, room temperature) was injected over about 30 seconds and discharged from the other tube (hereafter referred to as the "exhaust tube") fixed to the storage bag over about 30 seconds. The process from the start of injection to the end of discharge was performed over about 10 minutes. Then the same process was repeated. Next, 10 mL of vitrification solution (TCM199 solution containing 20% ethylene glycol, 20% DMSO, 10% carboxylated polylysine, 0.5 M sucrose, and 20% bovine serum, ice temperature) was poured from the injection tube over about 30 seconds. It was injected and discharged from the discharge pipe over about 30 seconds, and the process from the start of this injection to the end of discharge was performed over about 7 minutes. Then the same process was repeated. Thereafter, the storage bag was exposed to liquid nitrogen gas phase (approximately -150°C) to vitrify and freeze the cell sheet together with the storage bag.

The storage bag was placed on a heating plate (45°C) and a heating pack (38°C) was placed on the storage bag to rapidly thaw the cell sheet. Place the storage bag on the shaker again, pour 10 mL of the melted solution (TCM199 solution containing 1 M sucrose and 20% bovine serum, room temperature) from the injection tube over about 30 seconds, and drain the drain tube over about 30 seconds. The process from the start of injection to the end of discharge was performed over about 1 minute. Similarly, 10 mL of diluent (TCM199 solution containing 0.5 M sucrose and 20% bovine serum, room temperature) was injected over about 30 seconds and discharged from the discharge tube over about 30 seconds. The process took about 3 minutes. After that, 10 mL of the washing solution (TCM199 solution containing 20% bovine serum, room temperature) was injected over about 30 seconds and discharged from the discharge tube over about 30 seconds, and the process from the start of this injection to the end of discharge took about 5 minutes. went. Then the same process was repeated.

〔Experimental result〕
(1) Evaluation of cell sheet after vitrification freezing and thawing Evaluation of the cell sheet was performed from the following two viewpoints of recovery rate and cell survival rate.
Recovery rate: The shape (presence or absence of breakage) of cell sheets after vitrification and freezing was confirmed, and the percentage of sheets recovered without breakage was calculated.
Cell viability: Cells were dispersed by collagenase type II treatment and cell viability was determined by trypan blue staining.
For comparison, a cell sheet (non-vitrified section) that was not subjected to vitrification freezing and thawing treatment was used.

The results are shown in the table below. Fig. 4 shows morphological photographs of cell sheets after vitrification freezing and thawing (vitrification and thawing sections) and non-vitrification sections.

The morphology and cell viability of the cell sheet subjected to vitrification freezing and thawing treatment in the storage bag described above is equivalent to that of the cell sheet not subjected to these treatments, and can be repeatedly injected into and discharged from the storage container. It was found that the necessary liquid replacement was achieved by

(2) Liquid Replacement Efficiency Equilibrium liquid, vitrification liquid, melting liquid, diluent, and washing liquid were poured into storage containers in this order, and the equilibrium liquid, vitrification liquid, and washing liquid after perfusion or washing were collected, respectively. The liquid before injection was defined as the undiluted solution, and the liquid after recovery was defined as the recovered liquid. The results are shown in the table below.
The osmotic pressures of the undiluted solution and the recovered solution were measured using a freezing point depression osmometer (Osmomat030) and diluted with MQ as necessary.

The osmotic pressure of the recovery solution of the equilibrium solution, vitrification solution, and washing solution is a value that approximates the osmotic pressure of the original solution, and is similar to the osmotic pressure of the previously used solution (cell sheet culture solution, equilibrium solution, and vitrification solution). were completely different values. From this, it was found that the liquid exchange was performed almost completely.

Since the container of the present invention can be used to store biological samples such as cell sheets, the present invention can be used in industrial fields that handle such biological samples.

1: bag 2: opening 3: injection pipe 4: discharge pipe 5: injection port 6: discharge port 7: small hole

Claims (4)

Frozen cells or frozen spheroids embedded in a gelatin gel characterized by being prepared by a method comprising the following steps (1) to (3);
(1) a step of immersing a gelatin gel in which cells or spheroids are embedded in an equilibrium solution;
(2) a step of immersing the gelatin gel in a vitrification solution after step (1);
(3) After step (2), cooling the gelatin gel to freeze the cells or spheroids. Frozen cells or frozen spheroids embedded in gelatin gel according to claim 1, characterized in that the gelatin gel is a plate-like gelatin gel. The gelatin gel according to claim 1 or 2, wherein the gelatin gel has a substantially rectangular shape, and has a thickness of 0.5 to 5 mm, a long side of 10 to 200 mm, and a short side of 5 to 150 mm. frozen cells or frozen spheroids. The vitrification liquid contains ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose, the concentration of ethylene glycol is 10-25% by weight, the concentration of dimethyl sulfoxide is 10-25% by weight, and the concentration of carboxylated polylysine is 10-25% by weight. Frozen cells or frozen spheroids embedded in gelatin gel according to any one of claims 1 to 3, characterized in that the concentration is 1 to 15% by weight and the concentration of sucrose is 0.1 to 2M. .

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