Biological sample storage container

Abstract

To provide means for solving a problem of breakage seen in a conventional vitrification freezing preservation method of a biological sample.SOLUTION: A method for freezing a biological sample including the following processes (1) to (3): (1) a process of immersing gel in which a biological sample is embedded and gel supported by a mesh into equilibrium liquid; (2) a process of immersing the gel into vitrification liquid after process (1); and (3) process of cooling the gel after process (2) to freeze the biological sample.SELECTED DRAWING: None

Description

Technical Field

[0001] 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.

Background Art

[0002] Cell sheets are being widely applied in regenerative medicine. However, since culturing takes time, a time lag until transplantation is inevitable. For this reason, as a technique for storing cell sheets for a long period of time in a state where they can be used immediately when needed, a method of vitrifying and cryopreserving them has been developed (Non-Patent Document 1).

[0003] In the preservation method described in Non-Patent Document 1, the cell sheet is immersed in a equilibration solution, then immersed in a vitrification solution, and then frozen by exposing it to liquid nitrogen vapor. Also, thawing of the frozen cell sheet is performed 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.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] 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 its constituent cells. However, it has several problems. For example, in this method, immersion in each solution is performed by moving the cell sheet from one culture dish containing one immersion solution to another using tweezers or similar tools. Therefore, there is a risk of contamination by bacteria during the movement. Also, there is a risk of damaging the cell sheet by grasping it with tweezers. Furthermore, the operation requires skill, and there is a need for a new method that does not depend on the skill level of the operator. The inventor concluded that it is necessary to consider automating the cryopreservation method in order to solve these problems and to enable the cryopreservation of large quantities of cell sheets.

[0006] The present invention aims to solve the problems of conventional methods for vitrification and cryopreservation of cell sheets. [Means for solving the problem]

[0007] The inventors of the present invention have conducted extensive research to solve the above problems and have found that by placing a cell sheet in a resealable bag, installing two tubes with small holes around the periphery of the bag, injecting the immersion solution into the bag through one tube, and discharging the immersion solution through the other tube, all of the aforementioned problems related to hygiene, damage, and automation in the immersion operation can be solved, thus completing the present invention.

[0008] In other words, the present invention provides the following [1] to [6]. [1] A biological sample storage container comprising 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 an discharge member for discharging liquid from the inside of the container to the outside of the container, characterized in that the injection member and the discharge member are positioned such that a flow of liquid is generated between the injection member and the discharge member, and this flow of liquid comes into contact with the biological sample contained in the container body.

[0009] [2] The biological sample storage container according to [1], characterized in that the injection member and discharge member are tubes, a portion of these tubes is located inside the container body, and a plurality of small holes are provided in that portion.

[0010] [3] The biological sample storage container according to [1] or [2], characterized in that an injection member and a discharge member are installed on the periphery of the container body.

[0011] [4] A biological sample storage container according to any one of [1] to [3], characterized in that the biological sample is a cell sheet.

[0012] [5] A method for freezing a biological sample, characterized by comprising the following steps (1) to (4), (1) A step of placing a biological sample in the container body of a biological sample storage container described in any of [1] to [4], (2) A step of injecting the equilibrium solution 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 in which the vitrification solution is injected from the injection member into the container body and brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member. (4) A step of cooling the biological sample storage container and freezing the biological sample.

[0013] [6] A method for thawing a frozen biological sample, characterized by comprising the following steps (1) to (4), (1) A step of heating a biological sample storage container described in any of [1] to [4], in which a frozen biological sample is contained within the container body. (2) A step in which the melting solution is injected from the injection member into the container body and brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member. (3) The process of injecting the diluent from the injection member into the container body and bringing it into contact with the biological sample, and then discharging the liquid inside the container from the discharge member. (4) A step in which the cleaning solution is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member. [Effects of the Invention]

[0014] 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. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows an example of a biological sample storage container according to the present invention. [Figure 2] This figure shows another example of a biological sample storage container according to the present invention. [Figure 3] This figure shows the biological sample storage container used in the example. [Figure 4] These are morphological photographs of cell sheets after vitrification and thawing (A), and cell sheets that were not vitrified (B). [Modes for carrying out the invention]

[0016] 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 an discharge member for discharging liquid from the inside of the container to the outside of the container, characterized in that the injection member and the discharge member are positioned such that a flow of liquid is generated between the injection member and the discharge member, and this flow of liquid comes into contact with the biological sample contained in the container body.

[0017] The container body can be anything that can hold a biological sample, but a bag-shaped container is preferred. The bag-shaped container preferably has an opening for inserting and removing the biological sample, and this opening preferably can be opened and closed by a zipper or the like. A commercially available zippered storage bag can be used as this bag-shaped container. The container body is not limited to a bag-shaped container; for example, it may be a container in which a film is adhered to the top and bottom surfaces of a frame-like structure. The shape of the container body can be anything, but it is preferably rectangular for ease of installation of the injection and discharge members. The material of the container body can also be anything, but a transparent material with good thermal conductivity is preferred. If the container body is a bag-shaped container, the material of the container body is preferably 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. When the biological sample is a cell sheet and the container is a rectangular bag-shaped container, it is preferable that the rectangle has a long side of 30 to 350 mm and a short side of 10 to 100 mm.

[0018] The injection member can be any type of material that can inject liquid from outside the container into the container, but it is preferable that a portion of it be located inside the container body, and that portion (located inside the container body) has multiple small holes. In such a tube, liquid can be injected from outside the container body into the container by allowing the liquid to flow in from outside the container body and out through the small holes. The diameter of the tube is not particularly limited as long as it is within the range that allows liquid to be injected from outside the container into the container, but it is preferably 0.5 to 5 mm. The material of the tube is not particularly limited as long as it can inject liquid from outside the container into the container, but Teflon®, polyethylene, silicone, etc., are preferred. 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 portion located inside the container body. In this case, the spacing between the small holes is not particularly limited, but it is preferably 5 to 15 mm. The inner diameter of the small holes provided in the tube is also not particularly limited, but it is preferably 0.5 to 3 mm. The shape of the tube is not particularly limited and may be straight, bent, or curved. The tube may be branched, or it may be integrated with the 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. Also, 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. Furthermore, the diameter of the tube opening or the inner diameter of the small holes does not have to be uniform, and they may be formed so that the amount of liquid flowing out from each small hole is approximately equal.

[0019] The discharging member may be any member that can discharge liquid from the inside of the container to the outside of the container, but a part of it is located inside the container body, and it is preferably a tube provided with a plurality of small holes in the part (located inside the container body). In such a tube, by allowing the liquid to flow into the small holes and flow out of the container body, the liquid can be discharged from the inside of the container to the outside of the container. The diameter of the tube, the material of the tube, the shape of the tube, the position of the small holes, and the inner diameter of the small holes may be the same as those of the tube used as the injection member.

[0020] From the perspective of manufacturing efficiency, the injection member and the discharging member may be exactly the same, or they may be partially different in shape, material, etc.

[0021] The injection member and the discharging member are installed at positions where a liquid flow occurs between the injection member and the discharging member, and this liquid flow contacts the biological sample contained in the container body. Here, "a liquid flow occurs between the injection member and the discharging member" means that the liquid moves without staying between the injection member and the discharging member. The liquid flow may be substantially linear or may be curved within a range where the liquid does not stay.

[0022] By installing the injection member and the discharging member at positions where a liquid flow occurs between them, the exchange between the liquid already in the container body and the liquid to be newly injected can be carried out promptly. On the contrary, when the injection member and the discharging member are at positions where no liquid flow occurs between them, for example, in the case of a rectangular container often seen in a cell culture container (for example, the cell culture container described in JP-A-2008-22778715), when there is an injection port (injection member) and a discharge port (discharging member) adjacent to one side of the container, the newly injected liquid from the injection port will be mixed with the liquid originally in the container. Therefore, except immediately after the start of injection, the liquid 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, and thus it takes a very long time for the liquid exchange.

[0023] The storage container of the present invention is mainly used in vitrification cryopreservation, where the rapid exchange of liquids described above is extremely important. This is because, in vitrification cryopreservation, biological samples are sequentially immersed in several different liquids (e.g., equilibrium solution, vitrification solution, etc.). If the liquids are not exchanged promptly, the samples may end up being immersed for a long time in a mixture of the equilibrium solution and vitrification solution, rather than being immersed in either. On the other hand, when exchanging the liquid medium in a cell culture vessel, it is not necessary to exchange the new liquid medium for the old liquid medium immediately, as the new liquid medium may potentially harm the cells. Therefore, it is preferable to exchange the liquids over a certain period of time. Based on the above findings, in considering the storage container of the present invention, which aims to automate the vitrification cryopreservation of biological samples, the inventors newly discovered that it is preferable to position the injection member and the discharge member so that a liquid flow occurs between them. Therefore, positioning the injection member and the discharge member so that a liquid flow occurs between them is also preferable when using the storage container of the present invention in vitrification cryopreservation.

[0024] If the biological sample does not come into contact with the liquid flow generated between the injection member and the discharge member, the liquid surrounding the biological sample may not be replaced with new liquid. To prevent this from happening, the injection member and the discharge member are positioned so that the flow generated between the two members comes into contact with the biological sample contained in the container body.

[0025] When the injection member and discharge member are the tubes described above, it is generally preferable to install them at the periphery of the container body so that a liquid flow can be created between the two members, and this liquid flow can come into contact with the biological sample. Specifically, when the container body is a rectangular bag-shaped container, it is preferable to install the injection member and discharge member along opposite sides of the rectangle.

[0026] The container of the present invention is a container for storing biological samples, preferably a container for cryopreserving biological samples, and more preferably a container for vitrifying and cryopreserving biological samples. Here, "biological sample" refers to a sample of biological origin, such as cells, spheroids, tissues, organs, fertilized eggs, embryos, or parts thereof (e.g., sections). The biological sample may be of human origin or of animal origin other than humans. Examples of animals other than humans include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, and monkeys.

[0027] The biological sample to be preserved is not particularly limited, but preferably it is a cell sheet. Here, "cell sheet" refers to an aggregate of cells that have been adhered or aggregated to form a sheet. To date, 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, but in the present invention, cell sheets of any of these cells can be preserved.

[0028] The biological sample may be placed directly in the container, or it may be placed together with appropriate protective materials or scaffolding. Specifically, the biological sample may be embedded in a gel, enclosed in a capsule, fixed on a chip, or coated with a permeable membrane before being placed in the container.

[0029] Recently, a method has been reported for artificially constructing microscale fiber-shaped cell tissue by culturing a mixed solution of cells and collagen while it is flowing through a tiny tube (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.

[0030] The inventors have reported a method for vitrifying and freezing mouse and 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, approximately 20 to 40 biological samples (embryos) can be placed in one hollow fiber, but since the container of the present invention can accommodate a large number of hollow fibers, it is 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 liquid. By arranging them in this way, it is possible to suppress the hollow fibers from obstructing the flow of liquid.

[0031] By embedding the biological samples to be preserved together in a single plate-shaped gel and then placing it in the container body, it becomes possible to freeze and preserve many biological samples together. Furthermore, if the gel is a heat-melting gel such as gelatin, the biological samples can be easily removed from the gel by heating. Such plate-shaped gels can also be used for preservation in the container of the present invention. Since the biological samples to be preserved are embedded together in this plate-shaped gel, its size will be not much different from that of the container body. Specifically, for example, if the plate-shaped gel is roughly rectangular, its thickness will be 0.5 to 5 mm, its long side 10 to 200 mm, and its short side 5 to 150 mm.

[0032] The liquid 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 equilibrium solution, vitrification solution, thawing solution, diluent, washing solution, etc.

[0033] Next, an example of a biological sample storage container of the present invention will be described using Figure 1. This biological sample storage container consists of a rectangular bag 1, an injection tube 3, and an outlet tube 4. One of the short sides of the bag 1 is provided with an opening 2 that can be opened and closed by a zipper or the like, and the biological sample is placed inside the bag 1 through this opening 2. The injection tube 3 and the outlet tube 4 are fixed inside the bag 1 so as to be along the long side of the bag 1. One end of the injection tube 3 is inside the bag 1, but the other end is outside the bag 1. An injection port 5 is provided at this external end. Multiple small holes 7 are provided at equal intervals in the portion of the injection tube 3 that is located inside the bag 1. Similarly to the injection tube 3, one end of the outlet tube 4 is inside the bag 1, but the other end is outside the bag 1. An outlet port 6 is provided at this external end. Although not shown in this figure, multiple small holes 7 are also provided at equal intervals in the portion of the outlet tube 4 that is located inside the bag 1, similar to the injection tube 3.

[0034] When a biological sample is placed in this biological sample storage container and vitrified, the equilibrium solution and vitrification solution are injected sequentially from the inlet 5. The equilibrium solution injected from the inlet 5 passes through the injection tube 3, flows out through the small hole 7, and moves into the inside of the bag 1. The equilibrium solution 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, the biological sample is contained inside the bag 1, and the equilibrium solution that flows out from the small hole 7 of the injection tube 3 comes into contact with the biological sample before flowing into the small hole 7 of the discharge tube 4. This immerses the biological sample in the equilibrium solution necessary for vitrification. The equilibrium solution that flows into the small hole 7 of the discharge tube 4 passes through the discharge tube 4 and is discharged to the outside of the bag 1 from the discharge port 6. After the equilibrium solution is injected, the vitrification solution is injected from the inlet 5. The injected vitrification solution is discharged to the outside of the bag 1 from the discharge port 6, similar to the case of the equilibrium solution. This allows the biological sample to be immersed in the vitrification solution necessary for vitrification freezing. At this time, a flow of vitrification solution occurs between the small hole 7 of the injection tube 3 and the small hole 7 of the discharge tube 4. However, since the small holes 7 are provided throughout the entire portion of these tubes located inside the bag 1, the flow of vitrification solution occurs throughout the entire interior of the bag 1. Therefore, the equilibrium solution present inside the bag 1 is quickly discharged and replaced with the vitrification solution, minimizing the time the biological sample is immersed in the mixture of equilibrium solution and vitrification solution. Thus, by sequentially injecting the equilibrium solution and vitrification solution as described above, the same operation as in conventional vitrification freezing, which involves transferring the biological sample from a container containing the equilibrium solution to a container containing the vitrification solution, is performed.

[0035] Furthermore, when injecting and discharging equilibrium solution, vitrification solution, melting solution, diluent, washing solution, etc., the same type of liquid may be injected and discharged multiple times as needed.

[0036] Another example of the biological sample storage container of the present invention will be explained using Figure 2. This biological sample storage container, like the container shown in Figure 1, consists of a rectangular bag 1, an injection tube 3, and a discharge tube 4, but the injection tube 3 and the discharge tube 4 are integrated. Similar to the container shown in Figure 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 part fixed along one long side of the bag 1 and parts fixed along two short sides. The parts fixed along the two short sides branch off from the part fixed along one long side near the two corners where the long and short sides 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 discharge tube 4 is fixed along the other long side of the bag 1, and its two ends are outside the bag 1. Discharge ports 6 are provided at these ends. The discharge pipe 4 is connected to the injection pipe 3 near the two corners where its long and short sides intersect, thereby integrating the discharge pipe 4 with the injection pipe 3. Multiple small holes 7 are provided at equal intervals in the portions of the injection pipe 3 and discharge pipe 4 located inside the bag 1 (some of the small holes in the injection pipe 3 and discharge pipe 4 are not shown in the figure). In Figure 2, it is preferable that the injection pipe 3 and discharge pipe 4 are not connected inside the pipes, as this prevents the liquids in both pipes from mixing.

[0037] By sequentially injecting the equilibrium solution and vitrification solution into this biological sample storage container, the biological sample can be sequentially immersed in the equilibrium solution and vitrification solution, similar to the container shown in Figure 1. However, in this container, in addition to the liquid flow from the part fixed along the long side of the injection tube 3 to the discharge tube 4, there is also a liquid flow from the part fixed along the short side of the injection tube 3 to the discharge tube 4. Therefore, when switching the liquid being injected from the equilibrium solution to the vitrification solution, the equilibrium solution present inside the bag 1 can be discharged more quickly and replaced with the vitrification solution.

[0038] [2] Freezing method The present invention relates to a method for freezing biological samples, characterized by comprising the following steps (1) to (4). Steps (1), (2), (3), and (4) are performed in this order. Steps (2) and (3) may be performed multiple times.

[0039] In step (1), the biological sample is placed in the container body of the biological sample storage container described above.

[0040] The storage of biological samples can be carried out according to the container. For example, if the container has an opening that can be opened and closed by a zipper or the like, the biological sample can be stored through that opening.

[0041] In step (2), the equilibrium solution is injected from the injection member into the container body, brought into contact with the biological sample, and then discharged from the discharge member.

[0042] The equilibrium 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, the culture medium in which the biological sample was cultured, to which ethylene glycol and dimethyl sulfoxide have been added, can be used as the equilibrium solution. The concentration of ethylene glycol in the equilibrium solution can be 1 to 20% by weight, preferably 5 to 15% by weight. The concentration of dimethyl sulfoxide in the equilibrium solution can be 1 to 20% by weight, preferably 5 to 15% by weight. The culture medium to which ethylene glycol and dimethyl sulfoxide are added can be appropriately selected depending on the biological sample. Specific examples of culture media include DMEM medium, RPMI1640 medium, and TCM199 medium.

[0043] The temperature of the equilibrium solution can be the same as that used in known vitrification cryotherapy methods, for example, 0 to 30°C.

[0044] The time for which the equilibrium solution is in contact with the biological sample may be the same as the immersion time in the equilibrium solution in known vitrification freezing methods, for example, 1 to 30 minutes.

[0045] In step (3), the vitrification solution is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member.

[0046] The vitrification solution used may be the same as that used in known vitrification freezing methods. For example, a solution prepared by adding ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose to the culture medium in which the biological sample was cultured can be used as the vitrification solution. The concentration of ethylene glycol in the vitrification solution can be 10 to 25% by weight, preferably 15 to 20% by weight. The concentration of dimethyl sulfoxide in the vitrification solution can be 10 to 25% by weight, preferably 10 to 20% by weight. The concentration of carboxylated polylysine in the vitrification solution can be 1 to 15% by weight, preferably 5 to 10% by weight. The concentration of sucrose in the vitrification solution can be 0.1 to 2 M, preferably 0.5 to 1 M.

[0047] The temperature of the vitrification solution can be the same as that used in known vitrification freezing methods, for example, 0 to 37°C.

[0048] The time the vitrification solution is in contact with the biological sample can be the same as the immersion time in the vitrification solution in known vitrification freezing methods, for example, 1 to 30 minutes.

[0049] In step (4), the biological sample storage container is cooled and the biological sample is frozen.

[0050] Any method used in known vitrification freezing methods may be used to freeze the biological sample, but it is preferable to freeze the biological sample in the liquid nitrogen gas phase according to the method described in M. Maehara et al., BMC Biotechnology 13(58), 2013.

[0051] [3] Melting method The present invention relates to a method for thawing frozen biological samples, characterized by comprising the following steps (1) to (4). Steps (1), (2), (3), and (4) are performed in this order. Steps (2), (3), and (4) may be performed multiple times.

[0052] In step (1), the above-mentioned biological sample storage container, in which the frozen biological sample is contained within the container body, is heated.

[0053] The heating temperature and heating time can be the same as those used in known thawing methods after vitrification freezing (e.g., M. Maehara et al., BMC Biotechnology 13(58), 2013), for example, 20 to 45°C for 1 to 60 seconds.

[0054] In step (2), the melting solution is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member.

[0055] The thawing solution used may be the same as those used in known thawing methods after vitrification freezing. For example, the culture medium in which the biological sample was cultured, with sucrose added, can be used as the thawing solution. The concentration of sucrose in the thawing solution can be 0.1 to 2 M, preferably 0.3 to 1 M.

[0056] The temperature of the molten material can be the same as that used in known thawing methods after vitrification freezing, for example, 25 to 37°C.

[0057] The time the thawing solution is in contact with the biological sample can be the same as the immersion time in the thawing solution in known vitrification freezing thawing methods, for example, 0.5 to 3 minutes.

[0058] 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 inside the container is discharged from the discharge member.

[0059] The diluent used may be the same as those used in known post-vitrification freezing thawing methods. For example, the culture medium in which the biological sample was cultured, with sucrose added, can be used as the diluent. The concentration of sucrose in the diluent can be 0.1 to 2 M, preferably 0.3 to 1 M.

[0060] The temperature of the diluent can be the same as that used in known post-vitrification thawing methods, for example, 20 to 37°C.

[0061] The time the diluent is in contact with the biological sample can be the same as the immersion time in the diluent in known thawing methods after vitrification freezing, for example, 1 to 10 minutes.

[0062] In step (4), the cleaning solution is injected from the injection member into the container body, brought into contact with the biological sample, and then the liquid inside the container is discharged from the discharge member.

[0063] The washing solution used may be the same as those used in known post-vitrification freezing thawing methods. For example, the culture medium in which the biological sample was cultured can be used as the washing solution.

[0064] The temperature of the washing solution can be the same as that used in known post-vitrification thawing methods, for example, 20 to 37°C.

[0065] The time the washing solution is in contact with the biological sample can be the same as the immersion time in the diluent in known thawing methods after vitrification freezing, for example, 1 to 10 minutes.

[0066] [4] Method for freezing and thawing plate-shaped gels Methods for embedding biological samples such as cells and spheroids in gels and then vitrifying them have been known for some time. However, the gels to be frozen using such methods are very small, and no method for vitrifying large gels such as the plate-shaped gels described above was known. Therefore, a method for vitrifying such plate-shaped gels without placing them in the biological sample storage containers described above, and a method for thawing them, are novel methods, and the present invention includes such methods. Specifically, the present invention also includes a method in which the plate-shaped gel is sequentially immersed in an equilibrium solution and a vitrification solution before vitrification, and a method in which the vitrified plate-shaped gel is heated and then sequentially immersed in a thawing solution, a diluent, and a washing solution. [Examples]

[0067] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0068] [Experimental Method] (1) Preparation of cell sheets Primary cultured cells derived from the knee cartilage of Japanese white rabbits (CosmoBio Co., Ltd.) were seeded in temperature-responsive culture dishes (UpCell, CellSeed Co., Ltd.) and cultured in growth medium (DMEM / F12 medium containing 20% ​​FBS) at 37°C and 5% CO2. When the cells reached confluence, the growth medium was replaced with differentiation medium (RMI1640 medium containing 10% FBS and 100 μM L-ascorbic acid), and the culture was continued.

[0069] Two weeks after sowing, a single layer of cell sheets was formed. These sheets were then stacked to create a two-layer structure, and cultured for another week.

[0070] (2) Preparation of cell sheet storage bags Two tubes (made of Teflon, with an inner diameter of 1.7 mm) were inserted into a polyethylene zipper bag (110 x 85 mm, with a film thickness of 0.063-0.064 mm) so that one end protruded outside the bag. The inserted tubes were secured by heat sealing along the long side of the bag. Small holes with an inner diameter of 0.6-0.8 mm were made in the portion of the tubes inserted into the bag at intervals of 5-10 mm. The appearance of this cell sheet storage bag is shown in Figure 3.

[0071] (3) Freezing and thawing of cell sheets The zipper of the storage bag mentioned above was opened, and the cell sheet was placed inside the bag. The cell sheet was then sandwiched between two nylon meshes (50 x 50 mm, thread diameter 95 μm, mesh opening 140 μm) and stored in a protected state.

[0072] The storage bag was placed on a shaker, and 10 mL of equilibrium solution (TCM199 solution containing 10% ethylene glycol, 10% DMSO, and 20% bovine serum, at room temperature) was injected from one tube fixed to the storage bag (hereinafter referred to as the "injection tube") over approximately 30 seconds, and then discharged from the other tube fixed to the storage bag (hereinafter referred to as the "discharge tube") over approximately 30 seconds. This entire process, from the start of injection to the end of discharge, took approximately 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 approximately 30 seconds, and then discharged from the discharge tube over approximately 30 seconds. This entire process, from the start of injection to the end of discharge, took approximately 7 minutes. Next, the same process was repeated. Subsequently, the storage bags were exposed to liquid nitrogen gas (approximately -150°C), and the cell sheets, along with the storage bags, were vitrified and frozen.

[0073] 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 then placed back on the shaker, and 10 mL of thawing solution (TCM199 solution containing 1 M sucrose and 20% bovine serum, room temperature) was injected from the injection tube over approximately 30 seconds, and then discharged from the discharge tube over approximately 30 seconds. This entire process, from injection to discharge, took approximately 1 minute. Similarly, 10 mL of diluent (TCM199 solution containing 0.5 M sucrose and 20% bovine serum, room temperature) was injected over approximately 30 seconds, and then discharged from the discharge tube over approximately 30 seconds. This entire process, from injection to discharge, took approximately 3 minutes. Subsequently, 10 mL of washing solution (TCM199 solution containing 20% ​​bovine serum, room temperature) was injected over approximately 30 seconds, and then discharged from the discharge tube over approximately 30 seconds. This entire process, from injection to discharge, took approximately 5 minutes. Next, the same process was repeated.

[0074] [Experimental results] (1) Evaluation of cell sheets after vitrification freezing and thawing The cell sheets were evaluated from two perspectives: recovery rate and cell viability. Recovery rate: The shape of the cell sheets after vitrification (presence or absence of damage) was checked, 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. Furthermore, for comparison, cell sheets that were not subjected to vitrification freezing or thawing (non-vitrified group) were used.

[0075] The results are shown in the table below. Furthermore, morphological photographs of cell sheets after vitrification freezing and thawing (vitrified and thawed sections) and the non-vitrified section are shown in Figure 4.

[0076] [Table 1]

[0077] The morphology and cell viability of cell sheets subjected to vitrification freezing and thawing in the aforementioned storage bags were equivalent to those of cell sheets that were not subjected to these treatments, indicating that the necessary fluid exchange was achieved by repeatedly injecting and discharging the cells into the storage container.

[0078] (2) Liquid displacement efficiency The equilibrium solution, vitrification solution, fusion solution, diluent, and washing solution were poured into storage containers in this order, and the equilibrium solution, vitrification solution, and washing solution were collected after perfusion or washing. The liquid before injection was designated as the stock solution, and the liquid after collection was designated as the recovered solution. The osmotic pressure of each stock solution and recovered solution was measured to check whether the liquid exchange was performed correctly. The results are shown in the table below. The osmotic pressure of the stock solution and the recovered solution was measured using a freezing point depression osmometer (Osmomat030), and diluted with MQ as needed.

[0079] [Table 2]

[0080] The osmotic pressures of the recovered equilibrium solution, vitrification solution, and washing solution were close to those of the original solutions, and were completely different from the osmotic pressures of the previously used solutions (cell sheet culture medium, equilibrium solution, and vitrification solution). This indicates that the solution exchange was almost completely successful. [Industrial applicability]

[0081] Since the container of the present invention can be used for storing biological samples such as cell sheets, the present invention is applicable to industrial fields that handle such biological samples. [Explanation of symbols]

[0082] 1: Bag 2: Opening 3: Injection tube 4: Discharge pipe 5: Inlet 6: Outlet 7: Small hole

Claims

1. A method for freezing a biological sample, comprising the following steps (1) to (3): (1) immersing a gel in which a biological sample is embedded and which is supported by a mesh in an equilibration solution; (2) after step (1), immersing the gel in a vitrification solution; (3) After step (2), cooling the gel to freeze the biological sample.

2. 2. The method for freezing a biological sample according to claim 1, wherein the gel supported by the mesh is a gel sandwiched between the meshes.

3. 3. The method for freezing a biological sample according to claim 1, wherein the gel is a gelatin gel.

4. 4. The method for freezing a biological sample according to claim 1, wherein the biological sample is a cell or a spheroid.

5. 5. The method for freezing a biological sample according to claim 1, wherein the gel is a plate-shaped gel.

6. 6. The method for freezing a biological sample according to claim 1, wherein the gel is approximately rectangular, with 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.

7. 7. The method for freezing a biological sample according to claim 1, wherein the vitrification solution 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.

8. A gel in which a biological sample frozen by the method for freezing a biological sample according to any one of claims 1 to 7 is embedded.

9. A method for thawing a frozen biological sample, comprising the following steps (1) to (4): (1) a step of heating the gel according to claim 8; (2) after step (1), immersing the gel in a melting liquid; (3) after step (2), immersing the gel in a diluent; (4) After step (3), immersing the gel in a cleaning solution.

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