JP7668528B2 - Biological sample storage container - Google Patents

Description

Patent Law Article 30, Paragraph 2 applies Publisher: Japan Society for Regenerative Medicine Publication name: 16th General Meeting of the Japan Society for Regenerative Medicine Program and Abstracts, 405 pages Publication date: February 1, 2017 March 7-9, 2017 Presentation at the 16th General Meeting of the Japan Society for Regenerative Medicine

The present invention relates to a container for storing biological samples such as cell sheets, and a method for freezing and thawing biological samples using the container.

Cell sheets are being widely applied in regenerative medicine, but because the cultivation process takes time, a time lag before transplantation is unavoidable. For this reason, a method of vitrification and cryopreservation has been developed as a technique for long-term storage of cell sheets so that 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 equilibration 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 melting 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 technology 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 one immersion liquid to a culture dish containing another immersion liquid with tweezers or the like. Therefore, there is a risk of contamination with germs during the movement. In addition, there is a risk of the cell sheet being damaged by picking the cell sheet with tweezers. Furthermore, the operation requires skill, and a new method that does not depend on the skill of the operator is required. In order to solve these problems and to enable the cryopreservation of a large number of cell sheets, the inventors have come to the conclusion that it is necessary to consider automating the cryopreservation method.

The present invention aims to solve these problems associated with conventional cell sheet vitrification cryopreservation methods.

As a result of extensive research into solving the above problems, the inventors discovered that by placing a cell sheet in a sealable bag, placing two tubes with small holes on the periphery of the bag, injecting the soaking liquid into the bag through one tube and discharging the soaking liquid through the other tube, it is possible to solve all of the hygiene, breakage, and automation problems associated with the soaking operation described above, and thus completed the present invention.

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 liquid from outside the container into the inside of the container, and a discharge member for discharging liquid from inside the container to outside the container, characterized in that the injection member and the discharge member are positioned so that a flow of liquid is generated between the injection member and the discharge member, and such that this flow of liquid comes into contact with the biological sample contained in the container body.

[2] The biological sample storage container according to [1], characterized in that the inlet member and outlet member are tubes, parts of these tubes are located inside the container body, and the parts have multiple small holes.

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

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

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

[6] A method for thawing a frozen biological sample, comprising the following steps (1) to (4):
(1) a step of heating the biological sample storage container according to any one of [1] to [4], in which a frozen biological sample is accommodated in the container body; (2) a step of injecting a melting liquid into the container body from an injection member, bringing the melting liquid into contact with the biological sample, and then discharging the liquid in the container from a discharge member;
(3) injecting a diluent into the container body through the injection member, bringing the diluent into contact with the biological sample, and then discharging the liquid in the container through the discharge member;
(4) A step of injecting a washing liquid into the container body through the injection member, bringing the washing liquid into contact with the biological sample, and then discharging the liquid inside the container through the discharge member.

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

FIG. 1 is a diagram showing an example of a biological sample storage container of the present invention. FIG. 13 is a diagram showing another example of the biological sample storage container of the present invention. FIG. 1 is a diagram showing a biological sample storage container used in the examples. Photographs of the morphology of a cell sheet after vitrification and thawing (A) and a cell sheet that was not subjected to vitrification (B).

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 outside the container into the inside of the container, and a discharge member for discharging liquid from inside the container to outside the container, and is characterized in that the injection member and the discharge member are installed in positions where a flow of liquid is generated between the injection member and the discharge member and where this liquid flow comes into contact with the biological sample contained in the container body.

The container body may be any type capable of containing a biological sample, but a bag-shaped container is preferred. The bag-shaped container is preferably provided with an opening for inserting and removing the biological sample, and this opening is preferably openable and closable by a zipper or the like. A commercially available zippered storage bag can be used as the bag-shaped container. The container body is not limited to a bag-shaped container, and may be, for example, a container in which a film is adhered to the upper and lower surfaces of a picture-frame-shaped frame. The container body may be any shape, but is preferably rectangular in view of ease of installation of the inlet and outlet members. The container body may be made of any material, but is preferably transparent and has good thermal conductivity. When the container body is a bag-shaped container, the material of the container body is preferably, for example, low-density polyethylene, medium-density polyethylene, ultra-high molecular weight polyethylene, polyethylene-polytetrafluoroethylene copolymer, polyethylene-1,2-dichloroethane copolymer, etc. The size of the container body can be determined according to the biological sample to be contained. If the biological sample is a cell sheet and the container body is a rectangular bag-shaped 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 any member capable of injecting liquid from the outside of the container into the inside of the container, but is preferably a tube with a part located inside the container body and with multiple small holes in the part (located inside the container body). In such a tube, liquid can be injected from the outside of the container body into the inside of the container by flowing liquid in from the outside of the container body and letting the liquid flow out from the small holes. The diameter of the tube is not particularly limited as long as it is within a range in which liquid can be injected from the outside of the container into the inside of the container, but is preferably 0.5 to 5 mm. The material of the tube is not particularly limited as long as it is capable of injecting liquid from the outside of the container into the inside of the container, but is preferably Teflon (registered trademark), polyethylene, silicone, etc. The position of the small holes provided in the tube is not particularly limited, but it is preferable that the small holes are provided at equal intervals throughout the entire part located inside the container body. In this case, the interval between the small holes is not particularly limited, but is preferably 5 to 15 mm. The inner diameter of the small holes provided in the tube is also not particularly limited, but is preferably 0.5 to 3 mm. The shape of the tube is not particularly limited, and may be a straight shape, a broken line shape, or a curved shape. The tube may be branched, or may be integrated with a tube used as a discharge member. One end of the tube opens to the outside of the container body, and the other end usually opens to the inside of the container body. However, both ends may open to the outside of the container body. If the tube is branched, there will be three or more ends, and all of them may open to the outside of the container body, or only some of them may open to the outside of the container body. The 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 approximately equal.

The discharge member may be any member capable of discharging liquid from inside the container to outside the container, but is preferably a tube with a portion located inside the container body and with multiple small holes in that portion (located inside the container body). In such a tube, liquid can be discharged from inside the container to outside the container by flowing liquid into the small holes and allowing the liquid to flow out to the outside of the container body. The diameter, material, shape, position, and inner diameter of the small holes of the tube may be similar to those of the tube used as the injection member.

From the standpoint of manufacturing efficiency, the injection member and the discharge member may be exactly the same, but they may differ in some areas, such as shape or material.

The injection member and the discharge member are placed in a position where a liquid flow occurs between the injection member and the discharge member, and where this liquid flow comes into contact with the biological sample contained in the container body. 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 stagnation. The liquid flow may be substantially linear, or may be curved to the extent that the liquid does not stagnate.

By placing the injecting member and the discharge member in a position where a liquid flow occurs between them, it becomes possible to quickly replace the liquid in the container body with the new liquid to be injected. On the other hand, if the injecting member and the discharge member are in a position where no liquid flow occurs between them, for example, if the injecting port (injecting member) and the discharge port (discharge member) are adjacent to one side of a rectangular container, which is often found in cell culture containers (for example, the cell culture container described in JP 2008-22715), the new liquid injected from the injecting port will mix with the liquid originally in the container, so what is discharged from the discharge port is not the liquid originally in the container, but a mixture of the new liquid and the liquid originally in the container, except immediately after the start of injection. For this reason, it takes a very long time to exchange the liquid.

The storage container of the present invention is mainly used in the vitrification freezing method, in which the above-mentioned rapid liquid exchange is very important. This is because in the vitrification freezing method, the biological specimen is immersed in a number of different liquids (e.g., equilibrium liquid, vitrification liquid, etc.) in sequence, and if the liquid exchange is not performed quickly, the specimen will be immersed for a long time in a mixture of both liquids, rather than in the equilibrium liquid or vitrification liquid. On the other hand, when exchanging the liquid medium in a cell culture container, it is not necessary to exchange the new liquid medium with the old liquid medium quickly, and since the new liquid medium may be harmful to the cells, it is preferable to exchange the liquid over a certain amount of time. In considering the storage container of the present invention, one of the purposes of which is to automate the vitrification freezing of biological specimens based on the above findings, the inventors have newly discovered that it is preferable to install both the inlet member and the outlet member so that a liquid flow occurs between them. Therefore, installing both the inlet member and the outlet member so that a liquid flow occurs between them is also preferable when using the storage container of the present invention in the vitrification freezing method.

If the biological sample does not come into contact with the flow of liquid occurring between the inlet member and the outlet member, there is a possibility that the liquid around the biological sample will not be replaced with new liquid. To prevent this from happening, the inlet member and the outlet member are positioned so that the flow occurring between the two members comes into contact with the biological sample contained in the container body.

When the inlet member and outlet member are the above-mentioned tubes, it is usually preferable to place these members at the periphery of the container body, since this allows a liquid flow to occur between the two members and this liquid flow comes into contact with the biological sample. Specifically, when the container body is a rectangular bag-shaped container, it is preferable to place the inlet member and outlet member along opposite sides of the rectangle.

The container of the present invention is a container for storing 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 sample derived from an organism, such as a cell, a spheroid, a tissue, an organ, a fertilized egg, an embryo, or a part of these (e.g., a slice). The biological sample may be derived from a human or a non-human animal. Examples of non-human animals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, and monkeys.

The biological sample to be preserved is not particularly limited, but is preferably a cell sheet. Here, "cell sheet" refers to a collection of cells formed into a sheet by adhering or aggregating cells. To date, cell sheets of various cells, such as cartilage cells, corneal epithelial cells, skin cells, oral mucosa epithelial cells, bladder epithelial cells, cardiac muscle cells, and periodontal ligament cells, have been produced, and in the present invention, cell sheets of any of these cells can be preserved.

The biological sample may be contained in the container body as is, or may be contained together with an appropriate protective material or scaffolding material. Specifically, the biological sample may be contained in the container body after being embedded in a gel, encapsulated, fixed on a chip, or covered with a permeable membrane.

Recently, a method has been reported for artificially constructing microscale fibrous cell tissues by pouring a mixture of cells and collagen into a microtube, solidifying it, and culturing it (H. Onoe, et al., Nature Materials, Vol. 12, pp. 584-590, 2013). Such fibrous cell tissues 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). Such hollow fibers containing biological samples can also be stored in the container of the present invention. In the above method, approximately 20 to 40 biological samples (embryos) can be placed in one hollow fiber, but the container of the present invention can accommodate a large number of hollow fibers, making it possible to store a very large number of biological samples. When storing such hollow fibers, it is preferable to arrange the hollow fibers in the container body so that they are parallel to the flow of the liquid. By arranging them in this way, it is possible to prevent the hollow fibers from obstructing the flow of the liquid.

By embedding the biological samples to be preserved in a single plate-shaped gel and then storing it in the container body, it becomes possible to freeze and preserve many biological samples together. Furthermore, if the gel is a gel that melts when heated, such as gelatin, the biological samples can be easily removed from the gel by heating. Such plate-shaped gels can also be used as the preservation target of the container of the present invention. Since the biological samples to be preserved are embedded in this plate-shaped gel collectively, its size is not much different from that of the container body. Specifically, for example, if the plate-shaped gel is approximately rectangular, it 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.

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 an equilibration liquid, vitrification liquid, melting liquid, dilution liquid, or washing liquid.

Next, an example of the biological sample storage container of the present invention will be described with reference to FIG. 1. This biological sample storage container comprises a rectangular bag 1, an injection tube 3, and an exhaust 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 placed inside the bag 1 through this opening 2. The injection tube 3 and the exhaust tube 4 are fixed inside the bag 1 along the long side 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 equal intervals in the part of the injection tube 3 located inside the bag 1. Similarly to the injection tube 3, one end of the exhaust tube 4 is inside the bag 1, while the other end is outside the bag 1. An exhaust port 6 is provided at this external end. Although not shown in this figure, a plurality of small holes 7 are also provided at equal intervals in the part of the exhaust tube 4 located inside the bag 1, similar to the injection tube 3.

When a biological sample is placed in this biological sample storage container and vitrified and frozen, an equilibrium liquid and a vitrification liquid are sequentially injected from the injection port 5. The equilibrium liquid 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. The equilibrium liquid that flows out from the small hole 7 of the injection tube 3 does not stagnate, but forms a flow (arrow in the figure) and flows into the small hole 7 of the discharge tube 4. Although not shown in this figure, a biological sample is contained inside the bag 1, and the equilibrium liquid that flows out from the small hole 7 of the injection tube 3 flows into the small hole 7 of the discharge tube 4 after contacting the biological sample. This allows the biological sample required for vitrification and freezing to be immersed in the equilibrium liquid. The equilibrium liquid that flows into the small hole 7 of the discharge tube 4 passes through the discharge tube 4 and is discharged from the discharge port 6 to the outside of the bag 1. After the injection of the equilibrium liquid, the vitrification liquid is injected from the injection port 5. The injected vitrification liquid is discharged from the discharge port 6 to the outside of the bag 1, just like the equilibrium liquid. This allows the biological specimen to be immersed in the vitrification liquid required for vitrification freezing. At this time, a flow of vitrification liquid occurs between the small hole 7 of the injection tube 3 and the small hole 7 of the discharge tube 4, but since the small hole 7 is provided throughout the entire portion of these tubes located inside the bag 1, the flow of vitrification liquid occurs throughout the entire interior of the bag 1. Therefore, the equilibrium liquid present inside the bag 1 is quickly discharged and replaced with the vitrification liquid, so the time during which the biological specimen is immersed in the mixture of the equilibrium liquid and the vitrification liquid is minimized. Therefore, by sequentially injecting the above-mentioned equilibrium liquid and vitrification liquid, the biological specimen is transferred from a container containing the equilibrium liquid to a container containing the vitrification liquid, which is essentially the same operation as that performed in conventional vitrification freezing.

When injecting and discharging the equilibration liquid, vitrification liquid, melting liquid, dilution liquid, cleaning liquid, etc., the same type 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. 2. This biological sample storage container, like the container shown in FIG. 1, also consists of a rectangular bag 1, an injection tube 3, and an exhaust tube 4, but the injection tube 3 and the exhaust tube 4 are integrated. Like the container shown in FIG. 1, 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. The injection tube 3 consists of a part fixed along one long side of the bag 1 and a part fixed along the two short sides, and the part fixed along the two short sides branches off from the part fixed along one long side near the two corners where the long side and the short side intersect. The two ends of the part fixed along the long side of the injection tube 3 are outside the bag 1, and injection ports 5 are provided at these ends. The exhaust tube 4 is fixed along the other long side of the bag 1, and its two ends are outside the bag 1. These ends are provided with exhaust ports 6. The discharge pipe 4 is connected to the injection pipe 3 near the two corners where the long and short sides intersect, and thus the discharge pipe 4 is integrated with the injection pipe 3. The portions of the injection pipe 3 and the discharge pipe 4 located inside the bag 1 have a plurality of small holes 7 at equal intervals (parts of the injection pipe 3 and the small holes in the discharge pipe 4 are not shown in the figure). In addition, 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 vitrification liquid into this biological sample storage container, the biological sample can be sequentially immersed in the equilibrium liquid and vitrification liquid, as in the container shown in Figure 1. 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, there is also a flow of liquid from the part fixed along the short side of the injection tube 3 to the discharge tube 4, so when the liquid to be injected is switched from the equilibrium liquid to the vitrification liquid, the equilibrium liquid present inside the bag 1 can be more quickly discharged and replaced with the vitrification liquid.

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

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

The biological sample can be stored in a container according to the type of container. For example, if the container has an opening that can be opened and closed using a zipper or the like, the biological sample can be stored through that opening.

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

The equilibration solution used may be the same as that used in known vitrification freezing methods (e.g., M. Maehara et al., BMC Biotechnology 13(58), 2013). For example, a medium in which a biological sample has been cultured may be added with ethylene glycol and dimethyl sulfoxide to be used as the equilibration solution. The concentration of ethylene glycol contained in the equilibration solution may be 1 to 20% by weight, preferably 5 to 15% by weight. The concentration of dimethyl sulfoxide contained in the equilibration solution may be 1 to 20% by weight, preferably 5 to 15% by weight. The medium to which ethylene glycol and dimethyl sulfoxide are added may be appropriately selected depending on the biological sample. Specific examples of the medium include DMEM medium, RPMI1640 medium, and TCM199 medium.

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

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

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

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

The temperature of the vitrification liquid may be similar to 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 time for immersion in the vitrification liquid in known vitrification freezing methods, and may 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 known vitrification freezing methods, but it is preferable to freeze the biological sample in the liquid nitrogen vapor phase according to the method described in M. Maehara et al., BMC Biotechnology 13(58), 2013.

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

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

The temperature and time for heating may be similar to those used in known thawing methods after vitrification freezing (e.g., M. Maehara et al., BMC Biotechnology 13(58), 2013), and may be, for example, 20 to 45°C for 1 to 60 seconds.

In step (2), the melting liquid is injected into the container body through the injection member and brought into contact with the biological sample, after which the liquid in the container is discharged through the discharge member.

The melting liquid used may be the same as that used in known methods for melting after vitrification freezing. For example, a medium in which a biological sample has been cultured, to which sucrose has been added, may be used as the melting liquid. The concentration of sucrose contained in the melting liquid may be 0.1 to 2 M, preferably 0.3 to 1 M.

The temperature of the melting liquid may be similar to that used in known methods of melting after vitrification freezing, and may be, for example, 25 to 37°C.

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

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

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

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

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

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

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

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

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

[4] Method of freezing and melting a plate-shaped gel A method of embedding a biological sample such as a cell or a spheroid in a gel and vitrifying and freezing it has been known for some time. However, the gel to be frozen in this method is very small, and a method of vitrifying and freezing a large gel such as the plate-shaped gel described above has not been known. Therefore, a method of vitrifying and freezing such a plate-shaped gel without storing it in the biological sample storage container described above, and a method of melting it are novel methods, and the present invention also includes such methods. Specifically, the present invention also includes a method of immersing the plate-shaped gel in an equilibrium solution and a vitrification solution in sequence, and then vitrifying and freezing it, and a method of warming the vitrified and frozen plate-shaped gel and immersing it in a melting solution, a dilution solution, and a washing solution in sequence.

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 sheets Primary cultured cells derived from knee cartilage of Japanese white rabbits (Cosmo Bio) were seeded on a temperature-responsive culture dish (Upcell, CellSeed) and cultured in a proliferation medium (DMEM/F12 medium containing 20% FBS) at 37°C under ₅ % CO2. When the cells reached confluence, the proliferation medium was replaced with a differentiation medium (RMI1640 medium containing 10% FBS and 100 μM L-ascorbic acid), and the culture was continued.

Two weeks after seeding, a single layer of cell sheets was formed, which were then stacked to form a double layer and cultured for another week.

(2) Preparation of cell sheet storage bag Two tubes (made of Teflon, inner diameter 1.7 mm) were inserted into a polyethylene zippered bag (110 x 85 mm, film thickness 0.063-0.064 mm) with one end protruding outside the bag. The inserted tubes were fixed by heat sealing along the long side of the bag. Small holes with inner diameters of 0.6-0.8 mm were made at intervals of 5-10 mm in the parts of the tubes inserted into the bag. The appearance of this cell sheet storage bag is shown in Figure 3.

(3) Freezing and thawing of cell sheet The zipper of the storage bag was opened, and the cell sheet was placed 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 stored in a protected state.

The storage bag was placed on a shaker, and 10 mL of equilibration solution (TCM199 solution containing 10% ethylene glycol, 10% DMSO, and 20% bovine serum, at room temperature) was injected from one tube (hereinafter referred to as the "injection tube") fixed to the storage bag over a period of about 30 seconds, and discharged from the other tube (hereinafter referred to as the "discharge tube") fixed to the storage bag over a period of about 30 seconds. This process from the start of injection to the end of discharge was carried out over a period of about 10 minutes. Next, 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, at ice temperature) was injected from the injection tube over a period of about 30 seconds, and discharged from the discharge tube over a period of about 30 seconds. This process from the start of injection to the end of discharge was carried out over a period of about 7 minutes. Next, the same process was repeated. The storage bag was then exposed to liquid nitrogen vapor (approximately -150°C), and the cell sheet was vitrified and frozen 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 top of the storage bag to rapidly thaw the cell sheet. The storage bag was placed on the shaker again, and 10 mL of thawing solution (TCM199 solution containing 1 M sucrose and 20% bovine serum, at room temperature) was injected from the injection tube over approximately 30 seconds and discharged from the discharge tube over approximately 30 seconds. This process from the start of injection to the end of discharge took approximately 1 minute. Similarly, 10 mL of dilution solution (TCM199 solution containing 0.5 M sucrose and 20% bovine serum, at room temperature) was injected over approximately 30 seconds and discharged from the discharge tube over approximately 30 seconds. This process from the start of injection to the end of discharge took approximately 3 minutes. After that, 10 mL of cleaning solution (TCM199 solution containing 20% bovine serum, room temperature) was injected over approximately 30 seconds and discharged from the discharge tube over approximately 30 seconds. This process from the start of injection to the end of discharge took approximately 5 minutes. Next, the same process was repeated.

[Experimental Results]
(1) Evaluation of cell sheets after vitrification, freezing and thawing The cell sheets were evaluated from the following two perspectives: recovery rate and cell viability.
Recovery rate: The shape of the cell sheet after vitrification (presence or absence of damage) was confirmed, and the percentage of sheets that could be recovered without damage was calculated.
Cell viability: Cells were dispersed by collagenase type II treatment, and cell viability was determined by trypan blue staining.
As a comparison, a cell sheet that was not subjected to vitrification freezing and thawing treatment (non-vitrified group) was used.

The results are shown in the table below. Figure 4 shows morphological photographs of the cell sheets after vitrification, freezing and thawing (vitrified and thawed areas) and the non-vitrified area.

The morphology and cell viability of the cell sheets that had been subjected to vitrification freezing and thawing treatments in the above-mentioned storage bag were equivalent to those of cell sheets that had not been subjected to these treatments, and it was found that the necessary liquid replacement was achieved by repeatedly injecting and discharging the cells into the storage container.

(2) Fluid replacement efficiency The equilibrium fluid, vitrification fluid, melting fluid, dilution fluid, and washing fluid were injected into a storage container in that order, and the equilibrium fluid, vitrification fluid, and washing fluid were collected after perfusion or washing. The fluid before injection was called the original fluid, and the fluid after collection was called the collected fluid. The osmotic pressure of each original fluid and collected fluid was measured to check whether the fluid replacement was normal. The results are shown in the table below.
The osmotic pressure of the original solution and the recovered solution was measured using a freezing point depression osmometer (Osmomat030) by diluting with MQ as necessary.

The osmotic pressure of the recovered equilibration solution, vitrification solution and washing solution was close to that of the original solution, and was completely different from the osmotic pressure of the solutions used previously (cell sheet culture solution, equilibration solution and vitrification solution). This demonstrated that the solution exchange had been almost completely carried out.

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

1: Bag 2: Opening 3: Inlet pipe 4: Outlet pipe 5: Inlet 6: Outlet 7: Small hole

Claims (4)

A method for freezing a biological sample, comprising the following steps (1) to (3):
(1) a step of immersing a gel plate having a biological sample embedded therein and supported by a mesh in an equilibrium solution;
(2) after step (1), immersing the gel in a vitrification liquid;
(3) after step (2), cooling the gel and freezing the biological sample;
And,
The biological sample is a spheroid, a tissue, an organ, a fertilized egg, an embryo, or a slice thereof;
The above method, wherein the gel is approximately rectangular, 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. The method for freezing a biological sample according to claim 1, characterized in that the gel supported by the mesh is a gel sandwiched between the meshes. The method for freezing a biological sample according to claim 1 or 2, characterized in that the gel is a gelatin gel. 4. The method for freezing a biological sample according to claim 1, wherein the vitrification liquid contains ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose, and the concentration of the ethylene glycol is 10 to 25% by weight, the concentration of the dimethyl sulfoxide is 10 to 25% by weight, the concentration of the carboxylated polylysine is 1 to 15% by weight , and the concentration of the sucrose is 0.1 to 2M.