JP2020156523A - Biological sample storage container - Google Patents

Abstract

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

Description

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

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

In the storage method described in Non-Patent Document 1, the cell sheet is immersed in an equilibrium solution, then immersed in a vitrifying solution, and then frozen by exposure to liquid nitrogen vapor. Further, the frozen cell sheet is thawed by heating the cell sheet on an electric heating plate and then immersing the thawed solution, the diluted solution, and the washing solution in this 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 cell sheets and high viability of constituent cells. However, there are some problems. For example, in this method, the immersion operation in each solution is performed by moving the cell sheet from the culture dish containing one immersion solution to the culture dish containing another immersion solution by tweezers or the like. Therefore, there is a risk that germs may be mixed in during movement. In addition, the cell sheet may be damaged by pinching the cell sheet with tweezers. Further, skill is required for the operation, and a new method that does not depend on the skill level of the worker is required. The inventor has come to the conclusion that it is necessary to consider automation of the cryopreservation method in order to solve these problems and to enable cryopreservation of a large amount of cell sheets.

An object of the present invention is to solve the problem of such a conventional method for vitrifying and cryopreserving a cell sheet.

As a result of diligent studies to solve the above problems, the present inventor puts the cell sheet in a sealable bag, installs two tubes having small holes on the peripheral edge of the bag, and installs one tube. We have found that by injecting the dipping solution into the bag through the bag and discharging the dipping solution through the other tube, all of the above-mentioned hygiene problems, breakage problems, and automation problems in the dipping operation can be solved. It came to be completed.

That is, the present invention provides the following [1] to [6].
[1] A biological sample storage container having a container body for accommodating a biological sample, an injection member for injecting a liquid from the outside of the container to 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 for injection. The member and the discharge member are installed at a position where a liquid flow is generated between the injection member and the discharge member, and the liquid flow comes into contact with the biological sample contained in the container body. A biological sample storage container characterized by being

[2] The injection member and the discharge member are pipes, and a part of these pipes is located inside the container body, and a plurality of small holes are provided in the pipes [1]. The described biological sample storage container.

[3] The biological sample storage container according to [1] or [2], wherein the injection member and the discharge member are installed on the peripheral edge 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 method for freezing a biological sample, which comprises the following steps (1) to (4).
(1) A step of accommodating a biological sample in the container body of the biological sample storage container according to any one of [1] to [4].
(2) A process in which 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.
(3) A step of injecting the vitrified 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 for thawing a frozen biological sample, which comprises 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 the frozen biological sample is housed in the container body (2) The melt is injected from the injection member to the container body. The process of injecting into a container, contacting it with a biological sample, and then discharging the liquid inside the container from the discharge member,
(3) A step of injecting a diluted solution from an injection member into a container body, bringing it into contact with a biological sample, and then discharging the liquid inside the container from the discharge member.
(4) A step of injecting a cleaning liquid from an injection member into a container body, bringing it into contact with a 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 the biological sample using the container.

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

Hereinafter, the present invention will be described in detail.
[1] Biological sample storage container The biological sample storage container of the present invention includes a container body for accommodating a biological sample, an injection member for injecting a liquid from the outside of the container into the container, and a discharge for discharging the liquid from the inside of the container to the outside of the container. A biological sample having a member, the injection member and the discharge member at a position where a liquid flow is generated between the injection member and the discharge member, and the liquid flow is housed in the container body. It is characterized in that it is installed at a position where it comes into contact with.

The container body may be any one that can accommodate a biological sample, but a bag-shaped container is preferable. The bag-shaped container is preferably provided with an opening for taking in and out a biological sample, and the opening is preferably openable and closable by a zipper or the like. As this bag-shaped container, a commercially available storage bag with a zipper can be used. The container body is not limited to the bag-shaped container, and may be, for example, a container in which a film is adhered to the upper surface and the lower surface of the frame-shaped frame. The shape of the container body may be any shape, but it is preferably rectangular in terms of ease of installation of the injection member and the discharge member. The material of the container body may be any material, but a transparent material having good thermal conductivity is preferable. When the container body is a bag-shaped 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 or the like is preferable. 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 body is a rectangular bag-shaped container, the rectangular shape is preferably 30 to 350 mm on the long side and 10 to 100 mm on the short side.

The injection member may be any one as long as it can inject liquid from the outside of the container into the inside of the container, but a part of the injection member is located inside the container body and the portion (located inside the container body). Is preferably a tube provided with a plurality of small holes. In such a pipe, 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 flowing out from the small holes. The diameter of the tube is not particularly limited as long as the liquid can be injected from the outside of the container to 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 it can inject a liquid 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 pipe are not particularly limited, but it is preferable that the small holes are provided at equal intervals over the entire portion located inside the container body. In this case, the distance between the small holes is not particularly limited, but is preferably 5 to 15 mm. The inner diameter of the small hole provided in the pipe is not particularly limited, but is preferably 0.5 to 3 mm. The shape of the pipe is not particularly limited, and may be a linear shape, a polygonal linear shape, or a curved shape. The pipe may be branched or integrated with the pipe used as a discharge member. One of the ends of the tube opens outside the container body and the other end usually opens inside the container body. However, both ends may be open to the outside of the container body. Further, when the pipe is branched, the end is 3 or more, but all of them may be opened to the outside of the container body, or only a part may be opened to the outside of the container body. Further, the diameter of the pipe 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 equal.

The discharge member may be any material as long as it can discharge the liquid from the inside of the container to the outside of the container, but a part of the discharge member is located inside the container body and the portion (located inside the container body). Is preferably a tube provided with a plurality of small holes. In such a pipe, 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 holes and flowing out to the outside of the container body. The diameter of the pipe, the material of the pipe, the shape of the pipe, the position of the small hole, and the inner diameter of the small hole may be the same as those of the pipe used as the injection member.

The injection member and the discharge member may be exactly the same from the viewpoint of manufacturing efficiency, but the shape, material, and the like may be partially different.

The injection member and the discharge member are installed at a position where a liquid flow is generated between the injection member and the discharge member, and the liquid flow comes into contact with the biological sample contained in the container body. Has been done. Here, "a flow of liquid occurs between the injection member and the discharge member" means that the liquid moves between the injection member and the discharge member without staying. The flow of the liquid may be substantially linear or curved as long as the liquid does not stay.

By installing the injection member and the discharge member at a position where a liquid flow occurs between them, it is possible to quickly exchange the liquid contained in the container body with the liquid to be newly injected. Will be. On the other hand, when the injection member and the discharge member are in a position where no liquid flow occurs between them, for example, a cell culture container (for example, a cell culture container described in JP-A-2008-22715). ), Etc., when there is an injection port (injection member) and an discharge port (discharge member) adjacent to one side of the rectangular container, the new liquid injected from the injection port is originally inside the container. Since it will be mixed with a certain liquid, it is not the liquid originally in the container, but the liquid that is a mixture of the new liquid and the liquid originally in the container, except immediately after the start of injection. .. Therefore, it takes a very long time to change the liquid.

The storage container of the present invention is mainly used in the vitrification freezing method, but in the vitrification freezing method, the above-mentioned rapid liquid exchange is very important. This is because in the vitrification freezing method, the biological sample is sequentially immersed in a plurality of different liquids (for example, equilibrium liquid, vitrification liquid, etc.), but if the liquids are not exchanged promptly, for example, the equilibrium liquid. This is because it is not soaked in the vitrified liquid, but is soaked in a mixture of both liquids for a long time. On the other hand, when replacing the liquid medium in the cell culture container, it is not necessary to replace the new liquid medium with the old liquid medium promptly, and the new liquid medium may harm the cells. It is preferable that the liquid is exchanged over a certain period of time. Based on the above findings, in examining the storage container of the present invention for the purpose of automating the vitrification cryopreservation of biological samples, the inventors have found that a flow of liquid flows between the injection member and the discharge member. It was newly found that it is preferable to install both members so as to occur. Therefore, it is preferable to install both members so that a liquid flow is generated between the injection member and the discharge member, even when the storage container of the present invention is used in the vitrification freezing method.

This happens because if the biological sample does not come into contact with the flow of liquid that occurs between the injecting and discharging members, the liquid around the biological sample may not be exchanged for new liquid. The injection member and the discharge member are installed at positions where the flow generated between the two members comes into contact with the biological sample contained in the container body.

When the injection member and the discharge member are the above-mentioned pipes, usually, by installing these members on the peripheral edge of the container body, a liquid flow can be generated between the two members, and the liquid flow can be generated. Will come into contact with the biological sample, so it is preferable to install it in such a position. Specifically, when the container body is a rectangular bag-shaped container, it is preferable to install an injection member and a discharge member along opposite sides of the rectangle, respectively.

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 "biological sample" refers to a sample derived from an organism, such as cells, spheroids, tissues, organs, fertilized eggs, embryos, or a part thereof (for example, a section). The biological sample may be of human origin or of non-human animal origin. Examples of non-human animals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, monkeys and the like.

The biological sample to be stored is not particularly limited, but is preferably a cell sheet. Here, the "cell sheet" refers to an aggregate of cells formed into a sheet by adhering or aggregating cells. To date, various cell sheets such as chondrocytes, corneal epithelial cells, skin cells, oral mucosal epithelial cells, bladder epithelial cells, myocardial cells, and root membrane cells have been produced. The cell sheet of any of the cells 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 contained in a container body.

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

The present inventor has reported a method of vitrifying and freezing mouse and porcine embryos in hollow threads (H. Matsunari, et al., Journal of Reproduction and Development Vol. 58 (2012) No. 5 p. 599-608). The hollow thread containing such a biological sample can also be stored in the container of the present invention. In the above method, the number of biological samples (embryos) that can be put into one hollow thread is about 20 to 40, but since the container of the present invention can accommodate a large number of hollow threads, It is possible to store a large number of biological samples. When storing such hollow yarn, it is preferable to arrange the hollow yarn in the container body so as to be parallel to the flow of the liquid. By arranging in this way, it is possible to prevent the hollow yarn from obstructing the flow of the liquid.

By embedding the biological samples to be stored in one plate-shaped gel and then storing them in the container body, many biological samples can be cryopreserved together. Further, when the gel is a gel that is melted by heat such as gelatin, the biological sample can be easily taken out from the gel by heating. Such a plate-shaped gel can also be stored in the container of the present invention. Since the biological sample to be stored is collectively embedded in this plate-shaped gel, its size is not so different from that of the container body. 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, for example, equilibrium liquid, vitrification liquid, etc. A melt, a diluent, a cleaning solution, etc.

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 includes a rectangular bag 1, an injection tube 3, and a discharge 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 the opening 2. The injection pipe 3 and the discharge pipe 4 are fixed to the inside of 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 the outer end. A plurality of small holes 7 are provided at equal intervals in a portion of the injection pipe 3 located inside the bag 1. Like the injection pipe 3, the discharge pipe 4 has one end inside the bag 1, but the other end is outside the bag 1. A discharge port 6 is provided at the outer end. Although not shown in this figure, a plurality of small holes 7 are provided at equal intervals in a portion of the discharge pipe 4 located inside the bag 1 as in the injection pipe 3.

When a biological sample is placed in this biological sample storage container and vitrified and frozen, the equilibrium solution and the vitrified solution are sequentially injected from the injection port 5. The equilibrium liquid injected from the injection port 5 passes through the injection pipe 3, flows out from the small hole 7, and moves to the inside of the bag 1. The equilibrium liquid flowing out from the small hole 7 of the injection pipe 3 forms a flow (arrow in the figure) without staying, and flows into the small hole 7 of the discharge pipe 4. Although not shown in this figure, the biological sample is contained in the bag 1, and the equilibrium liquid flowing out from the small hole 7 of the injection tube 3 comes into contact with the biological sample and then is small in the discharge tube 4. It flows into the hole 7. As a result, the biological sample required for vitrification freezing is immersed in the equilibrium solution. 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 discharge port 6 to the outside of the bag 1. After injecting the equilibrium solution, the vitrified solution is injected through the injection port 5. The injected vitrified liquid is discharged from the discharge port 6 to the outside of the bag 1 as in the case of the equilibrium liquid. As a result, the biological sample required for vitrification freezing is immersed in the vitrification solution. At this time, a flow of the vitrifying liquid occurs between the small holes 7 of the injection pipe 3 and the small holes 7 of the discharge pipe 4, and the small holes 7 are provided in the entire portion of these pipes located inside the bag 1. Therefore, the flow of the vitrifying liquid occurs throughout the inside of the bag 1. Therefore, the equilibrium liquid existing inside the bag 1 is quickly discharged and replaced with the vitrified liquid, so that the time for the biological sample to be immersed in the mixed liquid of the equilibrium liquid and the vitrified liquid is minimized. It can be suppressed. Therefore, by sequentially injecting the above-mentioned 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 melting liquid, the diluting liquid, the cleaning liquid and the like, the same type of liquid may be injected and discharged a plurality of times as necessary.

Another example of the biological sample storage container of the present invention will be described with reference to FIG. Similar to the container shown in FIG. 1, this biological sample storage container also includes a rectangular bag 1, an injection pipe 3, and a discharge pipe 4, but the injection pipe 3 and the discharge pipe 4 are integrated. Similar to 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 side of the bag 1. The injection tube 3 is composed 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 is branched from the part fixed along one of the long sides near the two corners where the long side and the short side intersect. The two ends of the portion fixed along the long side of the injection tube 3 are outside the bag 1, and the injection port 5 is provided at these ends. The discharge pipe 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 in the vicinity of two corners where the long side and the short side intersect, 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 pipe 3 and the discharge pipe 4 located inside the bag 1 (a part of the injection pipe 3 and the small holes provided in the discharge pipe 4 are provided. 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 pipe because the liquids in both pipes do not mix with each other.

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

[2] Freezing Method The method for freezing a biological sample 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 a plurality of times.

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

The storage of the biological sample can be performed depending on the container. For example, when the container is provided with an opening that can be opened and closed by a zipper or the like, the biological sample can be accommodated through the opening.

In the 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 equilibrium 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 a biological sample has been cultured to which ethylene glycol and dimethyl sulfoxide have been added can be used as an equilibrium solution. The concentration of ethylene glycol contained in the equilibrium solution can be 1 to 20% by weight, preferably 5 to 15% by weight. The concentration of dimethyl sulfoxide contained in the equilibrium solution can be 1 to 20% by weight, preferably 5 to 15% by weight. The medium to which ethylene glycol or dimethyl sulfoxide is added can 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 solution may be the same as that used in the known vitrification freezing method, and may be, for example, 0 to 30 ° C.

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

In the step (3), the vitrified 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 the known vitrification freezing method. For example, a medium obtained by culturing a biological sample to which ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose are added can be used as a vitrification solution. The concentration of ethylene glycol contained in the vitrification solution can be 10 to 25% by weight, preferably 15 to 20% by weight. The concentration of dimethyl sulfoxide contained in the vitrification solution can be 10 to 25% by weight, preferably 10 to 20% by weight. The concentration of carboxylated polylysine contained in the vitrifying solution can be 1 to 15% by weight, preferably 5 to 10% by weight. The concentration of sucrose contained in the vitrification solution can be 0.1 to 2 M, preferably 0.5 to 1 M.

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

The time for contacting the vitrified solution with the biological sample may be the same as the immersion time in the vitrified solution in the known vitrification freezing method, and may be, for example, 1 to 30 minutes.

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

The method for freezing the biological sample may be any of the methods used in the known vitrification freezing method, but the liquid is according to the method described in M. Maehara et al., BMC Biotechnology 13 (58), 2013. It is preferable to freeze the biological sample in the nitrogen gas phase.

[3] Thawing Method The thawing method for a frozen biological sample of the present invention is characterized by including the 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 a plurality of times.

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

The heating temperature and heating time are similar to those used in known post-vitrification thawing methods (eg, M. Maehara et al., BMC Biotechnology 13 (58), 2013). Well, for example, it can be 1-60 seconds at 20-45 ° C.

In the step (2), the melt 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 thaw liquid used may be the same as that used in the known thaw method after vitrification freezing. For example, a medium in which a biological sample has been cultured to which sucrose has been added can be used as a melting solution. The concentration of sucrose contained in the melt can be 0.1 to 2 M, preferably 0.3 to 1 M.

The temperature of the melt may be similar to that used in the known thawing method after vitrification and freezing, and can be, for example, 25 to 37 ° C.

The time for contacting the melt with the biological sample may be the same as the time for immersion in the melt in the known thaw method after vitrification freezing, and can be, for example, 0.5 to 3 minutes.

In the step (3), the diluted solution 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 the known thawing method after vitrification freezing. For example, a medium obtained by culturing a biological sample to which sucrose is added can be used as a diluent. The concentration of sucrose contained in the diluent can be 0.1 to 2 M, preferably 0.3 to 1 M.

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

The time for contacting the diluent with the biological sample 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.

In the step (4), the cleaning 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 cleaning liquid used may be the same as that used in the known melting method after vitrification freezing. For example, the medium in which the biological sample was cultured can be used as a washing solution.

The temperature of the cleaning liquid may be the same as that used in the known melting method after vitrification freezing, and can be, for example, 20 to 37 ° C.

The time for contacting the washing liquid with the biological sample may be the same as the time for immersing 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 a plate-shaped gel A method of embedding a biological sample such as cells or spheroids in a gel and vitrifying and freezing it has been known for a long 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 above-mentioned plate-shaped gel has not been known. Therefore, the method of vitrifying and freezing such a plate-shaped gel without accommodating it in the above-mentioned biological sample storage container and the method of thawing it are novel methods, and the present invention is such a method. Also includes. Specifically, a method in which the above plate-shaped gel is sequentially immersed in an equilibrium solution and a vitrification solution and then vitrified and frozen, and a vitrified and frozen plate-shaped gel is heated to melt, dilute, and so on. The present invention also includes a method of sequentially immersing the glass in a cleaning solution.

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 (Upsell, CellSeed) and grown medium (DMEM / F12 containing 20% FBS). In the medium), the cells were cultured at 37 ° C. under 5% CO ₂ . 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 further culture was continued.

Since a monolayer cell sheet was formed 2 weeks after sowing, they were superposed to form a bilayer, and the cells were further cultured for 1 week.

(2) Preparation of cell sheet storage bag Two tubes (Teflon, inner diameter 1.7 mm) are placed in a polyethylene zipper bag (110 x 85 mm, film thickness 0.063-0.064 mm), and one end is outside the bag. I inserted it so that it would stick out. The inserted tube was fixed by a heat seal 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 inserted into the bag of the tube. The appearance of the cell sheet storage bag is shown in FIG.

(3) Freezing and thawing of the cell sheet The zipper of the above storage bag was opened, and the above cell sheet was placed inside the storage bag from there. The cell sheet was sandwiched between two nylon meshes (50 × 50 mm, thread diameter 95 μm, opening 140 μm) and housed in a state protected by these.

Place the storage bag on a shaker and from one tube fixed to the storage bag (hereinafter, this tube is referred to as the "injection tube") to the equilibrium solution (10% ethylene glycol, 10% DMSO, and 20% bovine). 10 mL of TCM199 solution containing serum (room temperature) is infused over about 30 seconds, and is discharged from the other tube fixed in the storage bag (hereinafter, this tube is referred to as "exhaust tube") over about 30 seconds. The process from the start of injection to the end of discharge was performed over 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, 0.5M sucrose, and 20% bovine serum, ice temperature) was applied from the injection tube over about 30 seconds. It was injected and discharged from the discharge pipe in about 30 seconds, and the process from the start of injection to the end of discharge was performed over about 7 minutes. Next, the same process was repeated. Then, the storage bag was exposed to a liquid nitrogen vapor phase (about -150 ° C.), and the cell sheet together with the storage bag was vitrified and frozen.

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, inject 10 mL of the melt (TCM199 solution containing 1 M sucrose and 20% bovine serum, room temperature) from the injection tube over about 30 seconds, and take about 30 seconds to drain the tube. The process from the start of injection to the end of discharge was carried out over about 1 minute. Similarly, 10 mL of diluted solution (TCM199 solution containing 0.5 M sucrose and 20% bovine serum, room temperature) is infused over about 30 seconds, discharged from the discharge tube over about 30 seconds, and from the start to the end of this injection. The process was carried out over about 3 minutes. After that, 10 mL of washing solution (TCM199 solution containing 20% bovine serum, room temperature) is injected over about 30 seconds, discharged from the discharge pipe over about 30 seconds, and the process from the start of injection to the end of discharge takes about 5 minutes. I went. Next, the same process was repeated.

〔Experimental result〕
(1) Evaluation of cell sheet after vitrification freezing and thawing The cell sheet was evaluated from the following two viewpoints of recovery rate and cell survival rate.
Recovery rate: The shape of the cell sheet after vitrification freezing (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.
In addition, as a comparison target, a cell sheet (non-vitrified plot) that was not subjected to vitrification freezing and thawing treatment was used.

The results are shown in the table below. Further, FIG. 4 shows a morphological photograph of the cell sheet (vitrified and thawed group) after vitrification freezing and thawing and the non-vitrified group.

The morphology and cell viability of the cell sheet subjected to the vitrification freezing and thawing treatment in the above storage bag are the same as those of the cell sheet not subjected to these treatments, and the injection and discharge into the storage container are repeated. It was found that the required liquid replacement was achieved.

(2) Liquid replacement efficiency The equilibrium solution, vitrified solution, melted solution, diluted solution, and cleaning solution were injected into a storage container in this order, and the equilibrium solution, vitrified solution, and cleaning solution after perfusion or cleaning were recovered. The liquid before injection was used as the undiluted solution, and the liquid after recovery was used as the recovered solution, and the osmotic pressures of each undiluted solution and the recovered solution were measured to check whether the liquids were exchanged normally. The results are shown in the table below.
The osmotic pressure of the stock solution and the recovered solution was measured by using a freezing point depression osmotic pressure meter (Osmomat030) and diluting with MQ as necessary.

The osmotic pressure of the recovered solution of the equilibrium solution, vitrification solution and washing solution is a value close to the osmotic pressure of the undiluted solution, and is the osmotic pressure of the solution (cell sheet culture solution, equilibrium solution and vitrification solution) used before that. Was a completely different value. From this, it was found that the liquid exchange was almost completely performed.

Since the container of the present invention can be used for storing a biological sample such as a cell sheet, the present invention can be used in an industrial field for handling such a biological sample.

1: Bag 2: Opening 3: Injection pipe 4: Discharge pipe 5: Injection port 6: Discharge port 7: Small hole

Claims (10)

A gel in which a frozen biological sample is embedded, which is produced by a method including the following steps (1) to (3).
(1) A step of immersing a gel in which a biological sample is embedded in an equilibrium solution.
(2) A step of immersing the gel in a vitrifying solution after the step (1).
(3) A step of cooling the gel and freezing the biological sample after the step (2). The gel in which the frozen biological sample according to claim 1, wherein the biological sample is a cell or a spheroid, is embedded. The gel in which the frozen biological sample according to claim 1 or 2 is embedded, wherein the gel is a plate-shaped gel. The frozen biological sample according to any one of claims 1 to 3, wherein the gel is substantially 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. Is an embedded gel. The vitrification solution contains ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose, and the concentration of ethylene glycol is 10 to 25% by weight, the concentration of dimethyl sulfoxide is 10 to 25% by weight, and the concentration of carboxylated polylysine is A gel in which the frozen biological sample according to any one of claims 1 to 4, wherein the concentration is 1 to 15% by weight and the concentration of sucrose is 0.1 to 2 M. A method for thawing a frozen biological sample, which comprises the following steps (1) to (4).
(1) A step of heating a gel in which a frozen biological sample according to any one of claims 1 to 5 is embedded.
(2) After the step (1), the step of immersing the gel in the melting liquid,
(3) A step of immersing the gel in a diluent after the step (2).
(4) A step of immersing the gel in a cleaning liquid after the step (3). A gel used for vitrification-freezing of a biological sample, wherein the biological sample is embedded in the gel. The gel according to claim 7, wherein the biological sample is a cell or a spheroid. The gel according to claim 7 or 8, wherein the gel is a plate-shaped gel. The gel according to any one of claims 7 to 9, wherein the gel is substantially 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.