JP2009148218A - Device for germ cells and method for cryopreservation of germ cells - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device suitable for handling germ cells, especially handling germ cells in cryopreservation or culture. <P>SOLUTION: The device 10 for germ cells is provided with a hollow fiber 11 opened at both ends, an injection needle 12 connected to an end of the hollow fiber 11 and forming a flow channel by continuing the inner space of the needle to the inner space of the hollow fiber 11, and a syringe 14 applying a suction pressure to the inner space of the injection needle 12. A germ cells 31 can be taken in the inner space of the hollow fiber 11 by applying suction pressure with the syringe 14. The hole of the hollow fiber 11 can pass a culture solution 32 and a vitrifying solution 41 while preventing the passage of the germ cells 31 and, accordingly, the culture liquid 32 in the inner space of the hollow fiber 11 and an intracellular fluid of the germ cells 31 can be substituted with the vitrifying solution 41 while keeping the state of the germ cells 31 included in the inner space of the hollow fiber 11. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a germ cell device that is suitably used for handling germ cells and can contain germ cells in the internal space of a hollow fiber.

  The present invention also relates to a germ cell cryopreservation method in which germ cells are contained in a hollow fiber and frozen.

  In the field of human infertility treatment and animal cloning, germ cells such as eggs and early embryos are sometimes cultured or cryopreserved. In this culture and cryopreservation, germ cells may be taken out of the medium or taken in and out of the cryopreservation container. Such a germ cell handling operation is performed by sucking germ cells into or discharging them from the straw using a straw-shaped instrument such as a pipette. In addition, a film-like or plate-like device (for example, Kitasato Supply Co., Ltd., Cryotop) is used, or a loop-shaped nylon thread (for example, Kitasato Supply Co., Ltd., Cryoloop) is used. Yes.

  Patent Document 1 discloses an instrument used for determining genetic information of an early embryo. This instrument is used to remove cells from an early embryo prior to transplantation and has a pipe material (2) similar to a pipette. This pipe material (2) is a thin glass tube, and a sharp piercing portion (3) is formed at the tip thereof. The other end of the pipe material (2) is connected to an operating instrument for micro-manipulators capable of micro-aspiration and discharge. The pipe material (2) is stabbed into the early embryo, and the cells in the early embryo are sucked into the pipe material (2).

  Patent Document 2 discloses an instrument for culturing or storing cells inside a hollow fiber. In this instrument, a bundle of hollow fibers (4) is fixed to a cell suspension flow tube (1) via a hollow fiber fixing portion (3), and the tip of the bundle of hollow fibers (4) is a curable resin ( 5). The cell suspension flow tube (1) is sucked with the bundle of hollow fibers (4) immersed in the medium, and the medium is filled into the bundle of cell suspension flow tubes (1) and hollow fibers (4). . Thereafter, when the cell suspension is injected into the bundle of hollow fibers (4) through the cell suspension flow pipe (1), cells are deposited inside the hollow fibers (4).

  Patent Document 3 discloses a method of seeding cells on a biological tissue matrix. In this method, a biodegradable hollow fiber filled with cells to be seeded is embedded in a biological tissue piece, and cells are released from the hollow fiber in the biological tissue piece.

JP 2003-33200 A JP 2003-339368 A JP 2007-168 A

  However, every time the medium is exchanged in the culture of germ cells, there is a problem that the operation of repeatedly taking out and discharging germ cells from the medium or the like using a conventional straw-shaped instrument is complicated.

  In addition, when cryopreserving germ cells, for example, if cryopreserving by vitrification, germ cells are sequentially immersed in a plurality of vitrification equilibrium solutions having a concentration gradient. There is a problem that the operation of repeatedly injecting and taking out germ cells from a plurality of types of vitrification equilibria using an instrument is complicated. Furthermore, the same complexity also becomes a problem when thawing cryopreserved germ cells and injecting them into the medium.

  The present invention has been made in view of these circumstances, and an object of the present invention is to provide a device suitable for handling germ cells and particularly suitable for handling germ cells during cryopreservation and culture. .

  Another object of the present invention is to provide means for easily cryopreserving germ cells.

  (1) A germ cell device according to the present invention is connected to a hollow fiber having both ends opened, and one end of the hollow fiber, and the internal space thereof becomes a flow path continuously with the internal space of the hollow fiber. A first tubular body, and a suction tool for applying a suction pressure to the internal space of the first tubular body.

  In the present invention, a germ cell is a comprehensive concept including primordial germ cells, sperm, sperm cells, eggs, oocytes, and early embryos. Here, the sperm cell refers to a progenitor cell in the process of becoming a sperm. An oocyte refers to a progenitor cell in the process of becoming an egg. An early embryo refers to a fertilized egg before implantation.

  The hollow fiber refers to a membrane in which a germ cell is formed in a straw shape that does not allow germ cells to pass through but has a plurality of pores with diameters that allow water, electrolytes, proteins, and the like to pass through. Examples of the membrane forming the hollow fiber include polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyethylene, polypropylene, cellulose acetate, and regenerated cellulose. The pore diameter of the membrane may be in the nanometer order. In addition, the thickness of the membrane is preferably 0.5 to 30 μm, and more preferably 1 to 10 μm, in order for the hollow fiber to be transparent enough to observe the aforementioned germ cells.

  The inner diameter of the hollow fiber is selected so that the target germ cells can be included, and is preferably 20 to 1000 μm, and more preferably 50 to 500 μm.

  The length of the hollow fiber can enclose several germ cells, and a length suitable for forming bubbles, which will be described later, is preferably 1 to 100 mm, and more preferably 10 to 40 mm. If the length of the hollow fiber is within this range, about 1 to several tens of cells can be encapsulated in the case of an oocyte, egg and early embryo, and several tens of cells in case of a primordial germ cell, sperm and sperm cell. About tens of thousands can be encapsulated, and bubbles can be formed on both open sides of the hollow fiber with respect to the encapsulated germ cells.

  The first tubular body can be connected to a hollow fiber and has rigidity to support the connected hollow fiber. The first tubular body can have a gas or liquid flow path in which the internal space is continuous with the internal space of the hollow fiber. The material of the first tubular body is not particularly limited, and examples thereof include metals such as stainless steel, plastics, and glass.

  The shape of the first tubular body is not particularly limited, and a circular tube, a square tube, or the like can be adopted. However, since the hollow fiber is generally cylindrical, a circular tube is preferable.

  The outer diameter or inner diameter of the first tubular body is selected in consideration of the connection with the hollow fiber. For example, if the hollow fiber is externally fitted to the first tubular body, the outer diameter of the first tubular body may be approximately the same as the inner diameter of the hollow fiber. Conversely, if the first tubular body is externally fitted to the hollow fiber, the inner diameter of the first tubular body may be approximately the same as the outer diameter of the hollow fiber. If the hollow fiber and the first tube are connected using a cylindrical joint, the outer diameter of the first tube may be approximately the same as the outer diameter of the hollow fiber.

  The length of the first tubular body is selected in consideration of handling, and about 50 to 1000 mm is preferable because it is excellent in handling.

  For the connection between the hollow fiber and the first tubular body, an internal fitting, an external fitting, or a mechanical connection structure by a joint may be selected, and further, adhesive fixing may be performed using a medical adhesive or the like.

  The suction tool is capable of sucking germ cells together with liquid from the opening of the hollow fiber using the hollow fiber and the internal space of the first tubular body as negative pressure. Examples of such a suction tool include a syringe barrel and a micro pump. In order to make the germ cell device disposable, it is preferable from the viewpoint of cost and disposal to select a disposable syringe as the suction tool.

  The connection between the suction tool and the first tubular body is not particularly limited. Even if the suction tool and the first tubular body are connected via a second tubular body as will be described later, the first tubular body is attached to the suction tool. It may be directly connected.

  When using the device for germ cells, for example, the germ cells in the medium are confirmed by a microscope, and the first tube of the device for germ cells is manipulated so that the hollow fiber connected to the first tube is in the medium. Insert the tip of the hollow fiber into the germ cell. Then, when the suction tool is operated to make the internal space of the hollow fiber negative pressure through the first tube, the negative pressure becomes the suction pressure, and germ cells together with the culture solution from the tip of the hollow fiber have the internal space of the hollow fiber. Sucked into. This operation is repeated when a plurality of germ cells are sucked into the inner space of the hollow fiber.

  When cryopreserving germ cells taken out from the medium, germ cells encapsulated in hollow fibers are immersed in a cryopreservation solution without removing germ cells from the hollow fibers. As described above, the hollow fiber hole does not allow germ cells to pass through, but allows a culture solution or a cryopreservation solution to pass through. Thus, while maintaining the state in which germ cells are encapsulated in the internal space of the hollow fiber, the hollow fiber The culture solution in the internal space of this cell or the intracellular solution of germ cells can be replaced with a cryopreservation solution. Thereby, a plurality of germ cells can be cryopreserved while being integrated with the hollow fiber.

  (2) The first tubular body and the suction tool may be connected by a flexible second tubular body.

  Examples of the flexible second tube include natural rubber, polyurethane, silicone rubber, polyvinyl chloride, polyethylene, polypropylene, ethylene / propylene copolymer and the like formed into a tube shape. The length of the second tube is not particularly limited, but if the length suitable for operating the first tube and the suction tool separately is adopted and is about 50 to 1000 mm, the first tube with one hand. It is preferable that the suction tool can be operated with the other hand while operating the body.

  Both ends of the second tube have a shape that can be connected to the first tube or the suction tool. The connection structure between the second tube body and the first tube body or the suction tool is not particularly limited, and may be directly connected by external fitting or indirectly through a joint or the like.

  Since the first tube body and the suction tool are connected by a flexible second tube body, the first tube body and the suction tool can be separately operated at positions separated from each other. Device handling is improved.

  (3) As the suction tool, a syringe having a port that communicates with the internal space, a gasket that is airtightly fitted into the internal space of the syringe and that can slide in the internal space, and the gasket connected to the gasket And a plunger extended in the sliding direction.

  Thereby, the suction tool can be made suitable for a disposable product from the viewpoint of cost and disposal. Further, the syringe and the plunger may be connected via a screwed portion. Thereby, there is an advantage that air or germ cell suspension is easily inhaled or discharged in and out of the hollow fiber.

  (4) In the germ cell cryopreservation method according to the present invention, a suction pressure is applied from one end of a hollow fiber that is open at both ends, and the germ cell and the second cell are separated from the germ cell suspension in which the germ cell floats in the first liquid. A first step of sucking one liquid from the other end of the hollow fiber into the internal space; a second step of immersing the hollow fiber containing germ cells and the first liquid in the internal space in a second liquid suitable for cryopreservation; A third step of cooling the hollow fiber containing germ cells in the internal space to freeze the germ cells.

  Germ cells to be cryopreserved float in the first liquid. The first liquid is a liquid suitable for suspending germ cells, and examples thereof include a culture solution. The culture medium is selected depending on the type and purpose of germ cells. For example, when culturing sperm cells, oocytes, or early embryos, TCM-199 medium, RPMI-1640 medium, DMEM medium, MEM medium, α-MEM Examples thereof include a medium and an IMDM medium. Moreover, HUCUM culture medium (made by Nipro) is mention | raise | lifted as a culture medium aiming at a human in-vitro fertilization. Examples of the medium for bovine in vitro fertilization include TCM-199 medium, SOF medium, CR1 medium, KSOM medium, IVF100 medium, IVF110S medium, IVMD101 medium, and IVD101 medium (manufactured by Functional Peptide Institute). Examples of media intended for in vitro fertilization of pigs include NCSU media and PZM-5 media (manufactured by Functional Peptide Institute).

  A germ cell suspension is one in which germ cells are suspended in the first liquid. Usually, a plurality of single-type germ cells are suspended in the first liquid.

  In the first step, for example, germ cells in the first fluid are confirmed with a microscope or the like, the hollow fiber is inserted into the cell suspension, and the tip of the hollow fiber is brought close to the germ cells. Then, a suction tool such as a syringe or a micropump is operated to apply a suction pressure to the internal space of the hollow fiber. If it does so, a germ cell will be attracted | sucked to the internal space of a hollow fiber with a 1st liquid from the front-end | tip of a hollow fiber. This operation is repeated when a plurality of germ cells are sucked into the inner space of the hollow fiber. Thereby, a desired number of germ cells and the first liquid are taken into the internal space of the hollow fiber.

  In the second step, the hollow fiber containing germ cells and the first liquid in the internal space is immersed in a second liquid suitable for cryopreservation. The second liquid is a liquid suitable for cryopreservation. As a method for cryopreserving germ cells, quick freezing in a high-concentration freezing protection solution and normal freezing in a low-concentration freezing protection solution can be mentioned. Rapid freezing in a high concentration cryoprotective liquid is also referred to as vitrification, and the frost damage protective liquid is also referred to as a vitrification solution. Examples of the intracellular protection type vitrification solution include dimethyl sulfoxide (DMSO) -containing liquid, ethylene glycol-containing liquid, and propanediol-containing liquid. Examples of the extracellular protection type vitrification solution include a sucrose-containing solution, a trehalose-containing solution, and a fill coal conley solution.

  To replace the first liquid contained in the internal space of the hollow fiber with the vitrification solution, the vitrification solution and the first liquid such as a culture solution are mixed as a second liquid at a ratio that provides an appropriate concentration gradient. A plurality of these may be used. That is, a plurality of types of second liquids in which the concentration of the vitrification solution gradually increases may be used. The more types of the second liquid having the concentration gradient, the more the first liquid contained in the inner space of the hollow fiber or the intracellular liquid of germ cells can be replaced with the vitrification solution under a slower condition. If the amount is too large, the operation becomes complicated, so 2 to 5 types of second liquids are usually used.

  When multiple types of second liquids are used, the hollow fiber is immersed in the second liquid having a low concentration of the vitrification solution. Then, replacement is performed for a predetermined time while stirring as necessary. As described above, the hole of the hollow fiber does not allow germ cells to pass but allows the first liquid and the second liquid to pass. Therefore, the internal space of the hollow fiber and the intracellular fluid of germ cells are replaced from the first liquid to the second liquid. At that time, germ cells are maintained in a state of being contained in the internal space of the hollow fiber. By performing this in order from the second liquid having a low concentration of the vitrification solution, the first liquid in the internal space of the hollow fiber, the intracellular fluid of the germ cells, and the like are replaced with the vitrification solution.

  In the third step, the hollow fiber containing the germ cell and the second liquid is cooled to freeze the germ cell in the hollow fiber. When the hollow fiber is taken out from the second liquid having the final concentration, germ cells and the second liquid are contained in the internal space of the hollow fiber. This hollow fiber is put into liquid nitrogen and cooled. Thereby, the germ cells contained in the internal space of the hollow fiber are frozen. Hollow fibers containing frozen germ cells are sealed in a container suitable for storage and stored in a freezer, liquid nitrogen, or liquid helium.

  As described above, germ cells that are contained in the internal space of the hollow fiber and cryopreserved are thawed by being immersed in a warming solution together with the hollow fiber. Thereafter, when a discharge pressure is applied from one end of the hollow fiber to the internal space, germ cells are discharged from the internal space of the hollow fiber to the outside. Further, if culturing is performed in a state where germ cells are included in the internal space of the hollow fiber, the hollow fiber including the germ cells may be put into a desired medium after thawing.

  (5) In the first step, a gas layer may be formed on both end sides of the germ cell and the first liquid sucked into the inner space of the hollow fiber.

  In the first step, when germ cells are sucked from the germ cell suspension into the hollow fiber internal space, only a small amount of the first liquid is sucked into the hollow fiber internal space prior to germ cell suction. Thereafter, the tip of the hollow fiber is taken out from the germ cell suspension, and a small amount of air is sucked into the internal space of the hollow fiber. Thereafter, the tip of the hollow fiber is again inserted into the germ cell suspension, and the germ cells and the first liquid are sucked into the internal space of the hollow fiber as described above. Thereby, in the internal space of the hollow fiber, a minute amount of air bubbles (gas layer) is formed on one opening side of the liquid layer in which germ cells and the first liquid exist.

  After the germ cells and the first liquid are sucked into the inner space of the hollow fiber, the tip of the hollow fiber is taken out from the germ cell suspension, and a small amount of air is sucked into the inner space of the hollow fiber. Thereafter, the tip of the hollow fiber is again inserted into the germ cell suspension, and only a small amount of the first liquid is sucked into the internal space of the hollow fiber. Thereby, in the internal space of the hollow fiber, a minute amount of bubbles (gas layer) is formed on the other opening side of the liquid layer in which germ cells and the first liquid exist.

  If gas layers are formed on both ends of the liquid layer where germ cells and the first liquid exist in the internal space of the hollow fiber, it becomes difficult for the liquid layer to easily move through the internal space of the hollow fiber. As a result, germ cells will not leak out from the internal space of the hollow fiber unless a suction pressure or discharge pressure is positively applied to the internal space of the hollow fiber.

  (6) In the first step, the other end of the hollow fiber is connected to the first tubular body, and the internal spaces continuously form a flow path, and suction pressure is applied to the internal space of the first tubular body. You may use the device for germ cells provided with the suction tool which provides.

  (7) In the second step, the hollow fiber connected to the first tubular body may be cut and the hollow fiber may be immersed in the second liquid.

  According to the germ cell device according to the present invention, each internal space is continuous as a flow path and the first tubular body and the hollow fiber are connected. Therefore, when suction pressure is applied to the flow path by the suction tool, Germ cells can be taken into the internal space of the hollow fiber. This hollow fiber hole does not allow germ cells to pass through, but allows culture medium or cryopreservation solution to pass through. Therefore, while maintaining the state in which germ cells are encapsulated in the hollow fiber interior space, The culture solution or the intracellular solution of germ cells can be replaced with a cryopreservation solution. That is, a device suitable for handling germ cells during cryopreservation and culture is realized.

  Further, according to the germ cell cryopreservation method according to the present invention, the germ cells are included in the internal space of the hollow fiber, and after that, the intracellular fluid of the germ cells is replaced with a cryopreservation solution and then frozen. The operation | work which takes in / out a germ cell with respect to several types of 2nd liquid provided with the concentration gradient is easy. In addition, since the cryopreserved germ cells are contained in the inner space of the hollow fiber, the thawing operation is easy, and after thawing, a plurality of germ cells are integrally contained in the inner space of the hollow fiber. Incubation can also be carried out in a fresh state. Therefore, germ cells can be easily frozen and stored, and further thawing and culturing can be facilitated.

  Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.

[Explanation of drawings]
FIG. 1 is a perspective view showing an overall configuration of a germ cell device 10 according to an embodiment of the present invention. 2 to 7 are schematic views showing the procedure of the germ cell cryopreservation method using the germ cell device 10. 8 and 9 are longitudinal sectional views showing the procedure of the germ cell cryopreservation method using the germ cell device 10. 2 to 8 show only a part of the hollow fiber 11 and the injection needle 12 of the germ cell device 10, and the tube 13 and the syringe barrel 14 are not shown in each drawing.

[Device 10 for germ cells]
As shown in FIG. 1, the germ cell device 10 mainly includes a hollow fiber 11, an injection needle 12, a tube 13, and a syringe barrel 14.

  The hollow fiber 11 is formed by forming a membrane having a plurality of pores having a diameter through which germ cells cannot pass through, but through which water, electrolytes, proteins, and the like can pass, into a straw shape having both ends opened. The inner diameter and the length of the hollow fiber 11 are appropriately selected according to the target germ cell, and the inner diameter is about several hundred μm and the length is several centimeters.

  The injection needle 12 is an embodiment of the first tubular body in the present invention. The injection needle 12 is a general one, and a stainless steel cannula is fixed to the hub. One end of the hollow fiber 11 is inserted into the end opening of the cannula of the injection needle 12, and the hollow fiber 11 and the injection needle 12 are fixed by a medical adhesive. Thereby, the internal space of the hollow fiber 11 continues to the internal space of the cannula of the injection needle 12. As will be described later, the continuous internal space is provided with a suction pressure and becomes a gas or liquid flow path.

  The tube 13 is an embodiment of the second tubular body in the present invention. The tube 13 is made of silicone rubber. The inner diameter and outer diameter of the tube 13 are appropriately selected according to the injection needle 12 and the syringe barrel 14. In FIG. 1, the tube 13 is shown with a part thereof omitted in the length direction. However, the length of the tube 13 is such that the operator separates the injection needle 12 and the syringe barrel 14 into both hands. The length is suitable for holding and operating, and is usually about several tens of centimeters. The flexibility of the tube 13 is suitable for the suction in which the tube 13 is not buckled and the inner space of the tube 13 is described later when the operator holds the syringe needle 12 and the syringe barrel 14 in both hands. When the negative pressure is increased, the tube 13 closes the internal space and does not collapse. One end of the tube 13 is connected to the hub of the injection needle 12 via a joint 15. Thereby, the internal space of the injection needle 12 is continuous with the internal space of the tube 13. Since this joint 15 may use a well-known thing suitably, detailed description is abbreviate | omitted here.

  The syringe barrel 14 is an embodiment of the suction tool in the present invention. In the syringe barrel 14, when the plunger 22 is pulled out with respect to the syringe 21, the gasket 23 connected to the tip of the plunger 22 moves to generate a negative pressure in the internal space of the syringe 21. This negative pressure is applied to the internal space of the injection needle 12 as a suction pressure.

  The configuration of the syringe barrel 14 is general. The syringe 21 has a substantially cylindrical shape, and a distal end side thereof is reduced in diameter to form a cylindrical attachment portion 24. Both ends of the attachment portion 24 are open, and one end communicates with the internal space of the syringe 21. That is, the internal space of the attachment portion 24 forms a port that connects the internal space of the syringe 21 and the outside. The other end of the tube 13 is externally fitted to the mounting portion 24, and the internal space of the syringe 21 and the internal space of the tube 13 are continuous. The base end side of the syringe 21 is opened, and the plunger 22 is inserted into the internal space from the base end side. The plunger 22 is longer than the length in the axial direction of the internal space of the syringe 21, and a part of the plunger 22 protrudes from the proximal end of the syringe 21 to the outside in a state where the plunger 22 is inserted to the innermost part of the internal space of the syringe 21. .

  A gasket 23 is connected to the tip of the plunger 22. The gasket 23 is a cylindrical body made of an elastic material such as rubber. The gasket 23 is airtightly fitted in the internal space of the syringe 21, and can slide along the plunger 22 along the internal space of the syringe 21 in the axial direction. The direction in which the plunger 22 extends coincides with the sliding direction of the gasket 23. When the gasket 23 is moved to the proximal end side of the syringe 21, the internal space of the syringe 21 becomes negative pressure, and gas or liquid is sucked from the attachment portion 24 into the internal space.

[Gem cell cryopreservation method]
The germ cell cryopreservation method using the germ cell device 10 will be described below. In the present embodiment, a technique using the germ cell device 10 is described as an example, but it goes without saying that an instrument other than the germ cell device 10 may be used in the germ cell cryopreservation method according to the present invention. Yes.

  The germ cell cryopreservation method described below mainly includes (S1) a first step of sucking the germ cells 31 and the culture solution 32 from the germ cell suspension into the internal space of the hollow fiber 11, and (S2) the germ cells 31. And a second step of immersing the hollow fiber 11 including the culture solution 32 in the vitrification solution 41, and a third step of cooling the hollow fiber 11 including the germ cell 31 and the vitrification solution 41 (S3). Has one step.

  Germ cells 31 to be cryopreserved are suspended in the culture solution 32. The culture solution 32 is held in a container such as a medium dish. However, in FIGS. 2 to 7, the container is omitted and only a plurality of germ cells 31 and a part of the culture solution 32 are shown. A plurality of germ cells 31 are suspended in the culture solution 32, and these germ cells 31 are of a single type collected from the same individual.

  As shown in FIG. 2, in the first step (S1), the injection needle 12 of the germ cell device 10 is operated with one hand, and the hollow fiber 11 is inserted into the culture solution 32. Then, the syringe barrel 14 is operated with the other hand, the plunger 22 is pulled out from the syringe 21, and a small amount of the culture solution 32 is sucked into the hollow fiber 11.

  After that, as shown in FIG. 3, the injection needle 12 is operated to take out the tip of the hollow fiber 11 from the culture solution 32, and the injection cylinder 14 is operated again to suck a small amount of air into the internal space of the hollow fiber 11. . Thereby, the trace amount culture solution 32 sucked into the hollow fiber 11 slightly moves in the inner space of the hollow fiber 11 to the injection needle 12 side.

  Thereafter, as shown in FIG. 4, the injection needle 12 is operated to insert the tip of the hollow fiber 11 into the culture solution 32 again, and while confirming the germ cells 31 in the culture solution 32 using a microscope, the hollow fiber 11 is hollow. The tip of the thread 11 is brought close to the germ cell 31. Then, the syringe barrel 14 is operated to suck the germ cells 31 together with the culture solution 32 into the internal space of the hollow fiber 11. Thereby, in the internal space of the hollow fiber 11, a trace amount of gas layer 34 is formed on the injection needle 12 side (one opening side) of the liquid layer 33 in which the germ cells 31 and the culture solution 32 are present. That is, in the inner space of the hollow fiber 11, a layer of only the culture solution 32, a gas layer 34, a germ cell 31, and a liquid layer 33 containing the culture solution 32 are formed in this order from the injection needle 12 side.

  Thereafter, as shown in FIG. 5, without removing the tip of the hollow fiber 11 from the culture solution 32, the tip of the hollow fiber 11 is brought close to another germ cell 31, and the syringe 14 is operated to move the inner space of the hollow fiber 11. The other germ cells 31 are aspirated together with the culture medium 32. The number of germ cells 31 sucked into the internal space of the hollow fiber 11 is not particularly limited, but in this embodiment, the three germ cells 31 are sucked into the internal space of the hollow fiber 11 together with the culture solution 32 to form the liquid layer 33. Is formed.

  After three germ cells 31 are sucked into the inner space of the hollow fiber 11, the tip of the hollow fiber 11 is taken out from the culture solution 32 by operating the injection needle 12 and the syringe cylinder 14 is operated as shown in FIG. Then, a small amount of air is sucked into the internal space of the hollow fiber 11. Thereby, the liquid layer 33 and the gas layer 34 in the hollow fiber 11 slightly move in the inner space of the hollow fiber 11 to the injection needle 12 side.

  Thereafter, as shown in FIG. 7, the injection needle 12 is operated to insert the tip of the hollow fiber 11 into the culture solution 32 again, and the syringe cylinder 14 is operated to enter the culture solution 32 into the internal space of the hollow fiber 11. Aspirate. Thereby, a very small amount of gas layer 35 is formed on the tip side (the other opening side) of the hollow fiber 11 with respect to the liquid layer 33.

  In the second step (S2), the hollow fiber 11 including the germ cells 31 and the culture solution 32 in the internal space is immersed in the vitrification solution 41. As described above, since the hollow fiber 11 is connected to the injection needle 12, as shown in FIG. 8, the injection needle 12 side of the hollow fiber 11 is cut to include the germ cell 31 and the culture solution 32. The hollow fiber 11 is separated from the germ cell device 10. Note that when the hollow fiber 11 is cut using scissors, air slightly enters the opening side of the hollow fiber 11 opposite to the cut portion, that is, the distal end side, due to the suck back action.

  As shown in FIG. 9, the vitrification solution 41 is adjusted in advance and injected into the container 50. When the culture solution 32 in the hollow fiber 11 is replaced with the vitrification solution 41, when it is desired to select a slow condition for the germ cells 31, the culture solution 32 has an appropriate concentration gradient as the vitrification solution 41. A plurality of mixed types are used. Such concentration gradients are set to 2 to 5 types, for example. When a plurality of types of vitrification solutions 41 are used, each preliminarily prepared vitrification solution 41 is poured into a plurality of containers 50.

  The container 50 includes a cylindrical main body 51 having an open end, and a cap 52 that is fitted to the main body 51 and seals the internal space of the main body 51. Such a container 50 is a general one. Since other configurations may be appropriately selected and used, detailed description thereof is omitted here.

  As shown in FIG. 9, the cut hollow fiber 11 is put into the container 50 into which the vitrification solution 41 has been injected. A plurality of germ cells 31 are included in the hollow fiber 11, and the hollow fiber 11 can be visually manipulated using tweezers. Then, if necessary, the container 50 is left for a predetermined time while being shaken, and the culture solution 32 in the hollow fiber 11 and the intracellular solution of the germ cells 31 are replaced with the vitrification solution 41. As described above, the hole of the hollow fiber 11 does not allow the germ cells 31 to pass through, but allows the culture solution 32 and the vitrification solution 41 to pass therethrough. Therefore, the culture solution 32 in the liquid layer 33 is replaced with the vitrification solution 41 while the germ cells 31 are held in the internal space of the hollow fiber 11. In addition, when substituting with the multiple types of vitrification solution 41 with which the concentration gradient was set, by replacing liquid sequentially about the thing from the density | concentration of the vitrification solution 41 high, the internal space of the hollow fiber 11 is carried out. The culture solution 32 is replaced with the vitrification solution 41.

  In the third step (S3), the hollow fiber 11 that has been replaced with the liquid is taken out of the container 50 using tweezers or the like, and is poured into liquid nitrogen to be cooled. Thereby, the germ cell 31 contained in the hollow fiber 11 with the vitrification solution 41 is vitrified. The hollow fiber 11 containing vitrified germ cells 31 is enclosed in a container suitable for cryopreservation and stored in a freezer.

  As described above, the germ cells 31 contained in the internal space of the hollow fiber 11 and cryopreserved are thawed by being immersed in a warming solution together with the hollow fiber 11. Thereafter, when a discharge pressure is applied from one end of the hollow fiber 11 to the internal space, the germ cells 31 are discharged from the internal space of the hollow fiber 11 to the outside. Moreover, if culture | cultivation is performed in the state where the germ cells 31 are included in the internal space of the hollow fiber 11, the hollow fiber 11 including the germ cells 31 may be put into a desired medium after thawing.

[Operational effects of this embodiment]
According to the aforementioned germ cell device 10, each internal space is continuous as a flow path and the injection needle 12 and the hollow fiber 11 are connected. Therefore, when suction pressure is applied to the flow path by the syringe 14, Germ cells 31 can be taken into the internal space of the hollow fiber 11. The hole of the hollow fiber 11 does not allow the germ cells 31 to pass through, but allows the culture solution 32 and the vitrification solution 41 to pass therethrough, so that while maintaining the state in which the germ cells 31 are encapsulated in the internal space of the hollow fiber 11, the hollow fibers The vitrification solution 41 can replace the culture solution 32 in the internal space 11 and the intracellular solution of the germ cells 31. Thus, the germ cell device 10 is suitable for handling the germ cells 41 during cryopreservation and culture.

  Further, since the injection needle 12 and the injection cylinder 14 are connected by a flexible tube 13, the injection needle 12 and the injection cylinder 14 can be operated separately at positions separated from each other. The handling of the device 10 is good.

  Further, as a suction tool according to the present invention, a syringe 21 having a port communicating with the internal space, a gasket 23 that is airtightly fitted into the internal space of the syringe 21 and that can slide in the internal space, and connected to the gasket 23. By using the syringe barrel 14 having the plunger 22 extended in the sliding direction of the gasket 23, it is advantageous from the viewpoint of cost and disposal when the germ cell device 10 is made a disposable product.

  Further, according to the aforementioned germ cell cryopreservation method, germ cells 31 are included in the internal space of the hollow fiber 11, and then the vitreous solution 41 is substituted for the intracellular solution of the germ cells 31 with the vitrification solution 41. Since it performs, the operation | work which takes in / out the germ cell 31 with respect to the multiple types of vitrification solution 41 in which the concentration gradient was provided is easy. In addition, since the cryogenically preserved germ cells 31 are included in the internal space of the hollow fiber 11, the thawing operation is easy. Furthermore, after thawing, a plurality of germ cells 31 are placed in the internal space of the hollow fiber 11. Culturing can also be carried out in the state of being integrally included. Therefore, the germ cells 31 can be easily vitrified and stored frozen, and further, the subsequent thawing and culturing operations are facilitated.

  Further, in the internal space of the hollow fiber 11, gas layers 34 and 35 are formed on both ends of the liquid layer 33 where the germ cells 31 and the culture solution 32 are present. However, the inner space of the hollow fiber 11 is not easily moved. Thereby, unless a suction pressure or discharge pressure is positively applied to the internal space of the hollow fiber 11, the germ cells 31 will not leak out from the internal space of the hollow fiber 11.

  In the present embodiment, the injection needle 12 and the injection cylinder 14 are connected by the tube 13, but in realizing the germ cell device according to the present invention, the injection needle 12 and the injection cylinder 14 are directly connected. Embodiments may be employed.

  Embryos from the 1 cell stage to the blastocyst stage were collected from BDF1 mice treated with superovulation. The embryos from the 1-cell stage to the blastocyst stage at each development stage were vitrified and thawed by the following procedure. In the following procedure, the basic medium is a HEPES buffered TCM-199 medium with 20% calf serum, and the equilibration liquid is 7.5% ethylene glycol and 7.5% as cryoprotectants. It is a liquid containing DMSO, the vitrification liquid is a liquid containing 15% ethylene glycol and 15% DMSO as cryoprotectants, and the melt is a liquid obtained by adding 1M sucrose to a basic medium.

[Working procedure]
(1) Using 20 germ cell devices 10 shown in FIG. 1, according to the procedures shown in FIG. 2 to FIG. Ten pieces were sucked into the hollow fiber 11 one by one. That is, for each developmental stage, five germ cell devices were used, and a total of 50 embryos were sucked into 10 hollow fibers 11 of 10 embryos in total.

(2) The hollow fiber 11 of the germ cell device 10 was cut as shown in FIG. Each hollow fiber 11 encapsulating the embryo was transferred into about 2 mL of equilibration solution and kept at room temperature for about 5 minutes.

(3) As shown in FIG. 9, the hollow fiber 11 immersed in the equilibrium solution was grasped with tweezers, moved to a vitrification solution previously cooled to an ice temperature, and held for about 1 to 2 minutes.

(4) The hollow fiber 11 immersed in the vitrification solution was grasped with tweezers, put into liquid nitrogen, and stored frozen.

(5) The hollow fiber 11 cryopreserved in liquid nitrogen was grasped with tweezers, moved into a melt previously warmed to 37 ° C., and held for 1 minute.

(6) The hollow fiber 11 immersed in the melt was grasped with tweezers, moved to a 0.5 M sucrose solution, and held for 3 minutes.

(7) The operation of grasping the hollow fiber 11 immersed in 0.5M sucrose solution with tweezers and moving it to a fresh basic medium is repeated three times, from the internal space of the hollow fiber 11 and the intracellular fluid of the germ cell 31 The cryoprotectant was completely removed.

[Survival judgment]
While holding one end of the hollow fiber 11 that has undergone the above-described operation procedure with tweezers, the hollow fiber 11 is gently handled using another tweezers, and the embryo contained in the inner space of the hollow fiber 11 is placed in a fresh culture solution. It was extruded and collected. The collected embryos were cultured for 24 hours using a commercially available embryo culture solution, and the survival was determined. In the case of a 1-cell stage embryo, a 2-cell stage embryo, and a morula stage embryo, those that developed to the 2-cell stage, 8-cell stage, and blastocyst stage were determined to be viable embryos. In addition, the blastocyst was determined to be viable when the blastocoele was reshaped after thawing. The results are shown in Table 1. As is clear from Table 1, the survival rate of all embryos was 100% regardless of the stage of embryo development.

FIG. 1 is a perspective view showing an overall configuration of a germ cell device 10 according to an embodiment of the present invention. FIG. 2 is a schematic diagram showing a procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 3 is a schematic diagram showing the procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 4 is a schematic diagram showing a procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 5 is a schematic diagram showing a procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 6 is a schematic diagram showing the procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 7 is a schematic diagram showing a procedure of a germ cell cryopreservation method using the germ cell device 10. FIG. 8 is a longitudinal sectional view showing the procedure of the germ cell cryopreservation method using the germ cell device 10. FIG. 9 is a longitudinal sectional view showing the procedure of the germ cell cryopreservation method using the germ cell device 10.

Explanation of symbols

10 ... Device for germ cells 11 ... Hollow fiber 12 ... Injection needle (first tube)
13 ... Tube 14 ... Syringe (suction tool)
21 ... Syringe 22 ... Plunger 23 ... Gasket 24 ... Mounting part (port)

Claims (7)

Hollow fibers open at both ends;
A first tubular body connected to one end of the hollow fiber, the internal space of which is a flow path continuous with the internal space of the hollow fiber;
A germ cell device comprising: a suction tool that applies suction pressure to the internal space of the first tubular body.   The device for germ cells according to claim 1, wherein the first tubular body and the suction tool are connected by a flexible second tubular body. The suction tool is
A syringe having a port leading to the internal space;
A gasket that is airtightly fitted into the internal space of the syringe and is capable of sliding the internal space;
The germ cell device according to claim 1, further comprising a plunger connected to the gasket and extending in a sliding direction of the gasket. Aspiration pressure is applied from one end of the hollow fiber that is open at both ends, and the germ cell and the first liquid are sucked from the other end of the hollow fiber into the internal space from the germ cell suspension in which germ cells float in the first liquid. A first step to:
A second step of immersing a hollow fiber containing germ cells and a first liquid in an internal space in a second liquid suitable for cryopreservation;
A germ cell cryopreservation method comprising: a third step of cooling a hollow fiber containing germ cells in an internal space to freeze the germ cells.   The germ cell cryopreservation method according to claim 4, wherein in the first step, a gas layer is formed on both sides of the germ cells sucked into the internal space of the hollow fiber and the first liquid.   In the first step, the other end of the hollow fiber is connected to the first tubular body, the internal spaces continuously form a flow path, and suction pressure is applied to the internal space of the first tubular body. The germ cell cryopreservation method according to claim 4 or 5, wherein a germ cell device provided with an aspirator is used.   The germ cell cryopreservation method according to claim 6, wherein in the second step, the hollow fiber connected to the first tubular body is cut, and the hollow fiber is immersed in the second liquid.

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