JP2019006695A - Biological sample preservation container - Google Patents

Abstract

To provide means that solves the hygienic problem, breakage problem, and automation problem found in the conventional biological sample vitrification cryopreservation method.SOLUTION: Provided is a biological sample preservation container including a container body for containing a biological sample, an injection member for injecting a liquid into the container from the outside of the container, and a discharge member for discharging a liquid from the inside of the container to the outside of the container and characterized in that the injection member and the discharge member are installed at such a position that a flow of liquid occurs between the injection member and the discharge member and the flow of the liquid contacts the biological sample contained in the container body.SELECTED DRAWING: Figure 1

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 a biological sample using the container.

  Although cell sheets are being widely applied to regenerative medicine, since time is required for culturing, a time lag until transplantation is inevitable. For this reason, a vitrified and cryopreserved method has been developed as a technique for preserving cell sheets for a long period of time in a ready-to-use state (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 vitrification solution, and then frozen by exposure to liquid nitrogen vapor. In addition, the frozen cell sheet is thawed by heating the cell sheet on an electric heating plate, and then immersing the melt, dilution solution, and washing solution in that order.

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

  The storage method described in Non-Patent Document 1 is an excellent technique that enables cryopreservation of a cell sheet while maintaining the structure of the cell sheet and the high survival rate of the constituent cells. However, there are some problems. For example, in this method, the immersion operation in each liquid is performed by moving the cell sheet from a culture dish containing a certain immersion liquid to a culture dish containing another immersion liquid using tweezers or the like. For this reason, there exists a possibility that various bacteria may mix in the case of a movement. In addition, the cell sheet may be damaged by picking the cell sheet with tweezers. Furthermore, a skill is required for the operation, and a new method that does not depend on the skill level of the worker is required. In order to solve these problems and to enable cryopreservation of a large amount of cell sheets, the inventor has thought that it is necessary to examine the automation of a cryopreservation method.

  The object of the present invention is to solve the problems of the conventional vitrification cryopreservation method for cell sheets.

  As a result of intensive studies in order to solve the above problems, the present inventor has stored a cell sheet in a sealable bag, installed two tubes having small holes in the peripheral edge of the bag, and one tube It was found that all of the above-mentioned hygiene problems, damage problems, and automation problems in the dipping operation can be solved by injecting the dipping liquid into the bag through the other tube and discharging the dipping liquid through the other tube. 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 main body that contains a biological sample, an injection member that injects liquid from the outside of the container into the container, and a discharge member that discharges liquid from the inside of the container to the outside of the container. 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 is in contact with a biological sample stored in the container body. A biological sample storage container characterized by comprising:

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

[3] The biological sample storage container according to [1] or [2], wherein the injection member and the discharge member are installed at 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 biological sample freezing method comprising 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) The step of injecting the equilibrium liquid from the injection member into the container body, contacting the biological sample, and then discharging from the discharge member;
(3) A step of injecting the vitrification liquid from the injection member into the container body and contacting 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, 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 the frozen biological sample is accommodated in the container body. (2) The molten liquid is supplied from the injection member to the container body. A step of discharging the liquid inside the container from the discharge member,
(3) a step of injecting the diluent from the injection member into the container body and contacting the biological sample, and then discharging the liquid inside the container from the discharge member;
(4) A step of discharging the liquid inside the container from the discharge member after injecting the cleaning liquid from the injection member into the container body and bringing it into contact with the biological sample.

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

It is a figure which shows an example of the biological sample storage container of this invention. 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 the Example. It is a form photograph of the cell sheet (A) after vitrification freezing and thawing, and the cell sheet (B) which did not perform 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 main body that contains a biological sample, an injection member that injects liquid from the outside of the container into the container, and a discharge that discharges liquid from the inside of the container to the outside of the container. A biological sample in which the injection member and the discharge member are positioned such that a liquid flow occurs between the injection member and the discharge member, and the liquid flow is accommodated in the container body It is characterized in that it is installed at a position where it touches.

  The container body may be anything as long as it can accommodate a biological sample, but a bag-like container is preferable. The bag-like container is preferably provided with an opening for taking in and out the biological sample, and this opening can be opened and closed by a zipper or the like. A commercially available storage bag with a zipper can be used as this bag-shaped container. The container body is not limited to a bag-shaped container, and may be a container in which a film is bonded to the upper and lower surfaces of a frame-like frame, for example. The shape of the container body may be any shape, but is preferably rectangular from the standpoint of easy installation of the injection member and the discharge member. The material of the container body may be any material, but a material that is transparent and has good thermal conductivity is preferable. When the container body is a bag-like container, the material of the container body is, for example, low density polyethylene, medium density polyethylene, ultrahigh molecular weight polyethylene, polyethylene-polytetrafluoroethylene copolymer, polyethylene-1,2- A dichloroethane copolymer is preferred. The size of the container body can be determined according to the biological sample to be accommodated. When the biological sample is a cell sheet and the container body is a rectangular bag-like container, the rectangle preferably has a long side of 30 to 350 mm and a short side of 10 to 100 mm.

  The injection member may be any member that can inject liquid from the outside of the container to the inside of the container, but a part thereof 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 tube, the liquid can be injected from the outside of the container to the inside of the container by flowing the liquid from the outside of the container body and flowing the liquid from the small hole. The diameter of the tube is not particularly limited as long as the liquid can be injected from the outside of the container 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 to the inside of the container, but Teflon (registered trademark), polyethylene, silicon and the like are preferable. The position of the small holes provided in the tube is not particularly limited, but the small holes are preferably provided at equal intervals throughout the portion 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 hole provided in the tube is not particularly limited, but is preferably 0.5 to 3 mm. The shape of the tube is not particularly limited, and may be a polygonal line shape or a curved shape in addition to a linear 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 of the tube usually opens to the inside of the container body. However, both ends may be open to the outside of the container body. Further, when the tube is branched, the ends are 3 or more, but all of them may be opened outside the container body, or only a part may be opened outside the container body. In addition, the diameter of the tube or the inner diameter of the small holes may not be uniform, and the flow amount of the liquid from each small hole may be formed to be substantially equal.

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

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

  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 is in contact with a biological sample stored in the container body. Has been. 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 flow of the liquid may be substantially linear, or may be 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, the liquid contained in the container body and the liquid to be newly injected can be quickly exchanged. It becomes like this. On the other hand, when the injection member and the discharge member are positioned so that no liquid flows between them, for example, a cell culture vessel (for example, a cell culture vessel described in JP-A-2008-22715) ) When there is an inlet (injection member) and an outlet (exhaust member) adjacent to one side of a rectangular container that is often seen, etc., the new liquid injected from the inlet originally enters the container Since it will be mixed with a certain liquid, the liquid discharged from the discharge port is not the liquid originally in the container except for immediately after the start of injection, but the liquid in which the new liquid and the liquid originally in the container are mixed. . 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 the vitrification freezing method, the above-described rapid liquid exchange is very important. This is because in the vitrification freezing method, a biological sample is sequentially immersed in a plurality of different liquids (for example, an equilibration liquid, a vitrification liquid, etc.). It is because it will be immersed in the liquid in which both liquids were mixed for a long time instead of being immersed in a vitrification liquid. On the other hand, when replacing the liquid culture medium in the cell culture container, it is not necessary to immediately replace the new liquid culture medium with the old liquid culture medium, and the new liquid culture medium may harm the cells. It is preferable to exchange the liquid over a certain amount of time. In examining the storage container of the present invention with the above knowledge as one of the purposes of automating the vitrification cryopreservation of biological samples, the inventors have found that the flow of liquid is 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.

  If the biological sample does not come into contact with the liquid flow generated between the injection member and the discharge member, this may occur because the liquid around the biological sample may not be replaced with a new liquid. In order to avoid this, the injection member and the discharge member are installed at a position where the flow generated between the two members comes into contact with the biological sample accommodated in the container body.

  When the injection member and the discharge member are the pipes described above, normally, 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 Since it comes to contact a biological sample, it is preferable to install in such a position. Specifically, when the container main body is a rectangular bag-shaped container, it is preferable to install the injection member and the discharge member respectively along the opposite sides of the rectangle.

  The container of the present invention is a container for storing a biological sample, preferably a container for storing a biological sample in a frozen state, and more preferably a container for storing a biological sample in a frozen state by vitrification. Here, the “biological sample” refers to a sample derived from a living organism such as a cell, a spheroid, a tissue, an organ, a fertilized egg, an embryo, or a part (for example, a slice) thereof. The biological sample may be derived from a human or an animal other than a human. Examples of animals other than humans 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. Various cell sheets such as chondrocytes, corneal epithelial cells, skin cells, oral mucosal epithelial cells, bladder epithelial cells, cardiomyocytes, periodontal ligament cells and the like have been prepared so far. The cell sheet of any of these cells can be a storage target.

  The biological sample may be accommodated in the container body as it is, but may be accommodated together with an appropriate protective material or scaffolding material. Specifically, a biological sample can be housed in a container body after being embedded in a gel, encapsulated in a capsule, fixed on a chip, or coated with a permeable membrane.

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

  The present inventor has reported a method of vitrifying mouse or pig embryos in a hollow fiber (H. Matsunari, et al., Journal of Reproduction and Development Vol. 58 (2012) No. 5 p. 599-608). A hollow fiber 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 placed in one hollow fiber is about 20 to 40, but the container of the present invention can accommodate a large number of hollow fibers, A very large number of biological samples can be stored. When storing such hollow fibers, the hollow fibers are preferably arranged in the container body so as to be parallel to the liquid flow. By arranging in this way, it is possible to suppress the hollow fiber from inhibiting the flow of the liquid.

  By embedding the biological samples to be stored together in one plate-like gel and then storing them in a container body, many biological samples can be stored frozen. Further, when the gel is a gel that melts by heat, such as gelatin, the biological sample can be easily removed from the gel by heating. Such a plate-like gel can also be stored in the container of the present invention. In this plate-like gel, biological samples to be stored are embedded together, so that the size thereof is not so different from that of the container body. Specifically, for example, when the plate-like gel is substantially rectangular, the thickness is 0.5 to 5 mm, the long side is 10 to 200 mm, and the short side is 5 to 150 mm.

  The liquid to be injected into the injection member is not particularly limited, but since the container of the present invention is mainly used for vitrification cryopreservation, the liquid is usually a liquid used for vitrification cryopreservation, such as an equilibrium liquid, a vitrification liquid, Melting liquid, dilution liquid, washing liquid, etc.

  Next, an example of the biological sample storage container of the present invention will be described with reference to FIG. The 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 put into the bag 1 from the opening 2. The injection tube 3 and the discharge 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 the outside end. A plurality of small holes 7 are provided at equal intervals in the portion of the injection tube 3 located inside the bag 1. Similarly to the injection tube 3, the discharge tube 4 has one end inside the bag 1 but the other end outside the bag 1. A discharge port 6 is provided at the outside end. Although not shown in this figure, a plurality of small holes 7 are also provided at equal intervals in the portion located inside the bag 1 of the discharge pipe 4 as in the case of the injection pipe 3.

  When a biological sample is placed in this biological sample storage container and frozen by vitrification, the equilibrium solution and the vitrification solution are sequentially injected from the injection port 5. The equilibrium liquid injected from the inlet 5 flows out of the small hole 7 through the injection tube 3 and moves into the bag 1. The equilibrium liquid flowing out from the small hole 7 of the injection pipe 3 does not stay and forms a flow (arrow in the figure) and flows into the small hole 7 of the discharge pipe 4. Although not shown in this figure, a biological sample is accommodated in the bag 1, and the equilibrium liquid that has flowed out from the small hole 7 of the injection tube 3 contacts the biological sample before the small sample in the discharge tube 4. It flows into the hole 7. Thereby, the biological sample necessary 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 vitrification solution 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 as in the case of the equilibrium liquid. Thereby, the biological sample required for vitrification freezing is immersed in the vitrification liquid. At this time, the vitrification liquid flows between the small hole 7 of the injection pipe 3 and the small hole 7 of the discharge pipe 4, and the small hole 7 is provided in the entire portion located inside the bag 1 of these pipes. As a result, the flow of vitrification liquid occurs throughout the interior of the bag 1. For this reason, since the equilibration liquid existing inside the bag 1 is quickly discharged and replaced with the vitrification liquid, the time during which the biological sample is immersed in the mixed liquid of the equilibration liquid and the vitrification liquid is minimized. It can be suppressed. Therefore, it is substantially the same as the operation performed in the conventional vitrification freezing in which the biological sample is transferred from the container containing the equilibrium liquid to the container containing the vitrification liquid by sequentially injecting the above-described equilibrium liquid and vitrification liquid. This is the same operation.

  In addition, in the injection and discharge of the equilibrium liquid, the vitrification liquid, the melting liquid, the dilution liquid, the cleaning liquid, and the like, the same kind 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 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 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 includes a portion fixed along one long side of the bag 1 and a portion fixed along two short sides, and is fixed along two short sides. The part which branches is branched from the part currently fixed so that one long side may be followed in the vicinity of two corner | angular parts where a long side and a short side cross. The two ends of the portion fixed along the long side of the injection tube 3 are outside the bag 1, and an injection port 5 is provided at these ends. The discharge pipe 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 in the vicinity of the 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 portion of the injection tube 3 and the discharge tube 4 located in the bag 1 (a small hole provided in a part of the injection tube 3 and the discharge tube 4 Not shown in the figure). In FIG. 2, it is preferable that the injection tube 3 and the discharge tube 4 are not connected inside the tube because the liquids in both the tubes are not mixed.

  By sequentially injecting the equilibrium liquid and the vitrification liquid into the biological sample storage container, the biological sample can be sequentially immersed in the equilibrium liquid and the vitrification liquid 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 tube 3 to the discharge tube 4, from the portion fixed along the short side of the injection tube 3. Since the liquid flow to the discharge pipe 4 also occurs, when the liquid to be injected is switched from the equilibrium liquid to the vitrification liquid, the equilibrium liquid present in the bag 1 is discharged more quickly and replaced with the vitrification liquid. Can do.

[2] Freezing method The method for freezing a biological sample of the present invention includes the 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 step (1), the biological sample is accommodated in the container body of the biological sample storage container.

  The biological sample can be accommodated 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 from the opening.

  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.

  The equilibration solution used may be the same as that used in the known vitrification freezing method (for example, M. Maehara et al., BMC Biotechnology 13 (58), 2013). For example, a medium in which a biological sample has been cultured and ethylene glycol and dimethyl sulfoxide added can be used as the equilibrium solution. The concentration of ethylene glycol contained in the equilibrium liquid can be 1 to 20% by weight, and preferably 5 to 15% by weight. The concentration of dimethyl sulfoxide contained in the equilibrium solution can be 1 to 20% by weight, and preferably 5 to 15% by weight. The medium to which ethylene glycol or dimethyl sulfoxide is added can be appropriately selected according to 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 a known vitrification freezing method, and can be set to, for example, 0 to 30 ° C.

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

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

  The vitrification liquid to be used may be the same as that used in the known vitrification freezing method. For example, what added ethylene glycol, dimethyl sulfoxide, carboxylated polylysine, and sucrose to the culture medium which culture | cultivated the biological sample can be used as vitrification liquid. The concentration of ethylene glycol contained in the vitrification solution can be 10 to 25% by weight, and preferably 15 to 20% by weight. The concentration of dimethyl sulfoxide contained in the vitrification solution can be 10 to 25% by weight, and preferably 10 to 20% by weight. The concentration of the carboxylated polylysine contained in the vitrification solution can be 1 to 15% by weight, and preferably 5 to 10% by weight. The concentration of sucrose contained in the vitrification solution can be 0.1 to 2M, and preferably 0.5 to 1M.

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

  The time for which the vitrification liquid is brought into contact with the biological sample may be the same as the immersion time in the vitrification liquid in the known vitrification freezing method, and 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 of freezing the biological sample may be any method used in the known vitrification freezing method, but according to the method described in M. Maehara et al., BMC Biotechnology 13 (58), 2013, a liquid is used. It is preferable to freeze the biological sample in a nitrogen gas phase.

[3] Thawing Method The thawing method of the 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 biological sample storage container in which the frozen biological sample is accommodated in the container body is heated.

  The warming temperature and warming time are the same as those used in the known thawing method after vitrification (eg, M. Maehara et al., BMC Biotechnology 13 (58), 2013). For example, it may be 1 to 60 seconds at 20 to 45 ° C.

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

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

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

  The time for bringing the melt into contact with the biological sample may be the same as the time for immersion in the melt in the known melting method after vitrification freezing, for example, 0.5 to 3 minutes.

  In step (3), the diluent is injected from the injection member into the container main body and 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 melting method after vitrification freezing. For example, a medium in which a biological sample has been cultured and sucrose added thereto can be used as a diluent. The density | concentration of the sucrose contained in a dilution liquid can be 0.1-2M, Preferably, it can be 0.3-1M.

  The temperature of the diluent 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 bringing the diluted solution into contact with the biological sample may be the same time as the immersion time in the diluted solution in the known melting method after vitrification freezing, and can be, for example, 1 to 10 minutes.

  In step (4), the cleaning liquid is injected from the injection member into the container main body and 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 and freezing. For example, a medium in which a biological sample has been 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 which the cleaning liquid is brought into contact with the biological sample may be the same time as the immersion time in the diluent in the known melting method after vitrification freezing, and can be, for example, 1 to 10 minutes.

[4] Method of freezing and thawing a plate-like gel A method of embedding a biological sample such as a cell or spheroid in a gel and freezing it by vitrification 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 a large gel such as the plate-like gel described above has not been known. Therefore, a method of freezing and freezing such a plate-like gel without containing it in the biological sample storage container and a method of thawing it are novel methods, and the present invention is such a method. Including. Specifically, after the above plate-like gel is sequentially immersed in the equilibrium solution and the vitrification solution, the method of freezing vitrification, and heating the vitrified plate-like gel, the melting solution, the dilution solution, In addition, a method of sequentially immersing in a cleaning solution is also included in the present invention.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these Examples.

〔experimental method〕
(1) Preparation of cell sheet Primary cultured cells derived from Japanese white rabbit knee cartilage (Cosmo Bio) are seeded in a temperature-responsive culture dish (Upsell, CellSeed) and growth medium (DMEM / F12 containing 20% FBS) Medium) at 37 ° C. under 5% CO ₂ . When the cells reached confluence, the growth medium was replaced with a differentiation medium (RMI1640 medium containing 10% FBS and 100 μM L-ascorbic acid), and the culture was further continued.

  Since a single-layer cell sheet was formed in 2 weeks after sowing, they were stacked to form a double layer and further cultured for 1 week.

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

(3) Freezing and thawing of cell sheet The zipper of the storage bag was opened, and the cell sheet was put into the storage bag from there. The cell sheet was sandwiched between two nylon meshes (50 × 50 mm, thread diameter 95 μm, mesh opening 140 μm) and stored in a protected state.

  Place the storage bag on a shaker, and equilibrate (10% ethylene glycol, 10% DMSO, and 20% bovine) from one tube (hereinafter referred to as “infusion tube”) fixed to the storage bag. Inject 10 mL of TCM199 solution containing serum (room temperature) over about 30 seconds, and drain it from the other tube (hereinafter referred to as “drain tube”) fixed to the storage bag over about 30 seconds. The process from the start of injection to the end of discharge was performed for 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) from the injection tube takes about 30 seconds. The injection was carried out from the discharge pipe over about 30 seconds, and the process from the start of injection to the end of discharge was carried out over about 7 minutes. Next, the same process was repeated. Thereafter, the storage bag was exposed to liquid nitrogen gas phase (about −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 the storage bag to rapidly thaw the cell sheet. Place the storage bag on the shaker again, inject 10 mL of the melting solution (1M sucrose and TCM199 solution containing 20% bovine serum, room temperature) from the injection tube over about 30 seconds, and discharge the tube over about 30 seconds. The process from the start of injection to the end of discharge was performed for about 1 minute. Similarly, inject 10 mL of diluent (0.5 M sucrose and TCM199 solution containing 20% bovine serum, room temperature) over about 30 seconds, discharge from the discharge tube over about 30 seconds, and start from the start of injection until the end of discharge. The process took about 3 minutes. Then, 10 mL of washing solution (TCM199 solution containing 20% bovine serum, room temperature) is injected over about 30 seconds, discharged from the discharge tube 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 viability.
Recovery: The shape (presence or absence of breakage) of the cell sheet after vitrification and freezing was confirmed, and the ratio of the sheet that was recovered without breakage was calculated.
Cell viability: Cells were dispersed by collagenase type II treatment, and cell viability was determined by trypan blue staining.
Moreover, the cell sheet (non-vitrification section) which does not perform vitrification freezing and a thawing process was used as a comparison object.

  The results are shown in the table below. In addition, FIG. 4 shows a morphological photograph of cell sheets after vitrification freezing and thawing (vitrification and thawing zone) and non-vitrification zone.

  The form and cell viability of the cell sheet that has undergone vitrification freezing and thawing treatment in the above storage bag is equivalent to the cell sheet that has not been subjected to these treatments, and repeated injection and discharge into the storage container. It was found that the necessary liquid replacement was achieved.

(2) Solution replacement efficiency The equilibration solution, vitrification solution, melting solution, dilution solution, and washing solution were poured into the storage container in this order, and the equilibration solution, vitrification solution, and washing solution after perfusion or washing were collected. The liquid before injection was used as a stock solution, and the liquid after collection was used as a collected solution. The osmotic pressure of each stock solution and the collected solution was measured, and it was examined whether the liquid was exchanged normally. The results are shown in the table below.
The osmotic pressure of the stock solution and the recovered solution was measured by diluting with MQ as necessary using a freezing point depression osmometer (Osmomat030).

  The osmotic pressure of the recovered solution of the equilibrium solution, vitrification solution and washing solution is similar to the osmotic pressure of the stock solution, and the osmotic pressure of the solution used before (cell sheet culture solution, equilibrium solution and vitrification solution) Were completely different values. 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 biological samples such as cell sheets, the present invention can be used in the industrial field in which such biological samples are handled.

1: Bag 2: Opening Portion 3: Injection Pipe 4: Discharge Pipe 5: Injection Port 6: Discharge Port 7: Small Hole

Claims (6)

  A biological sample storage container having a container main body for storing a biological sample, an injection member for injecting liquid from the outside of the container into the container, and a discharge member for discharging liquid from the inside of the container to the outside of the container. The member is installed at a position where a liquid flow is generated between the injection member and the discharge member, and the liquid flow is in contact with a biological sample stored in the container body. A biological sample storage container.   The living body according to claim 1, wherein the injection member and the discharge member are pipes, a part of the pipes is located inside the container body, and a plurality of small holes are provided in the part. Sample storage container.   The biological sample storage container according to claim 1, wherein the injection member and the discharge member are installed on a peripheral portion of the container main body.   The biological sample storage container according to any one of claims 1 to 3, wherein the biological sample is a cell sheet. A biological sample freezing method comprising the following steps (1) to (4):
(1) The process of accommodating a biological sample in the container main body of the biological sample storage container as described in any one of Claims 1 thru | or 4.
(2) The step of injecting the equilibrium liquid from the injection member into the container body, contacting the biological sample, and then discharging from the discharge member;
(3) A step of discharging the liquid inside the container from the discharge member after injecting the vitrification liquid from the injection member into the container body and bringing it into contact with the biological sample;
(4) A step of cooling the biological sample storage container and freezing the biological sample. A method for thawing a frozen biological sample, comprising the following steps (1) to (4):
(1) The step of heating the biological sample storage container according to any one of claims 1 to 4, wherein the frozen biological sample is accommodated in the container body (2) The molten solution is supplied from the injection member to the container body. A step of discharging the liquid inside the container from the discharge member,
(3) a step of injecting the diluent from the injection member into the container body and contacting the biological sample, and then discharging the liquid inside the container from the discharge member;
(4) A step of discharging the liquid inside the container from the discharge member after injecting the cleaning liquid from the injection member into the container body and bringing it into contact with the biological sample.