JP5062164B2 - Cell device and method for manufacturing cell device - Google Patents

Description

  The present invention relates to a cell device that can be suitably used for handling cells and can enclose cells in the internal space of a hollow fiber, and more particularly to a cell device having means suitable for preventing breakage of a hollow fiber.

  The present invention also relates to a method for producing a cell device using a core material suitable for preventing breakage of a hollow fiber as a jig suitable for coupling the hollow fiber to a tubular body.

  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 the object thereof is suitable for handling cells, and in particular, by using hollow fibers, it is suitable for handling cells during cryopreservation and culture, and An object of the present invention is to provide means suitable for preventing breakage of a hollow fiber.

  Another object of the present invention is to provide a means for easily producing a device for a cell using a core material suitable for preventing breakage of a hollow fiber as a jig suitable for coupling the hollow fiber to a tubular body. is there.

  (1) The cell device according to the present invention comprises a hollow fiber having both ends opened and a flow path connected to the first end of the hollow fiber, the inner space of which is continuous with the inner space of the hollow fiber. And a core material inserted through the hollow fiber. The core material can be removed from the hollow fiber in use.

  Examples of the cells in the present invention include germ cells. Germ cells are a generic concept that includes 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. Note that the cell device according to the present invention may be used for handling cells such as ES cells in addition to germ cells.

  The hollow fiber refers to a membrane in which a cell having a plurality of pores having a diameter that does not allow cells to pass through but allows water, electrolytes, proteins, and the like to pass through is formed into a straw shape. 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. Further, 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 have transparency that allows the aforementioned cells to be observed.

  The inner diameter of the hollow fiber is selected so that the target 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 cells, and a length suitable for forming bubbles to be described later is selected, preferably 1 to 100 mm, and more preferably 10 ~ 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, plastic, 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 core material can be inserted into and removed from the hollow fiber, and has a rigidity enough to support the hollow fiber so that the hollow fiber is not bent when inserted into the hollow fiber. The material of the core material is not particularly limited, and examples thereof include metals such as stainless steel and plastics. The core material may be inserted only through the hollow fiber or may be inserted through the hollow fiber and the first tubular body.

  The shape of the core material is not particularly limited, and a bar material such as a cylinder or a prism can be adopted. However, since the hollow fiber is generally cylindrical, it is preferably a columnar bar material. The outer diameter of the core material is selected in consideration of insertion into the hollow fiber, and an outer diameter slightly smaller than the inner diameter of the hollow fiber is preferable.

  When using the cell device, the core material is removed from the hollow fiber. Thereby, cells can be sucked into the hollow fiber. On the other hand, until the hollow fiber is supported by the core material, the hollow fiber is prevented from being bent due to an external force applied by the hollow fiber coming into contact with another member.

  After extracting the core material from the hollow fiber, for example, the cells in the medium are confirmed with a microscope or the like, and the first tubular body of the cell device is operated, so that the hollow fiber connected to the first tubular body is in the medium. And the tip of the hollow fiber is brought close to the cells. Then, when a negative pressure is applied to the inner space of the hollow fiber through the first tube using a suitable suction tool, the negative pressure becomes a suction pressure, and the cells are brought together with the culture solution from the tip of the hollow fiber into the hollow fiber. Sucked into space. This operation is repeated when a plurality of cells are sucked into the internal space of the hollow fiber.

  When cryopreserving the cells removed from the medium, the cells encapsulated in the hollow fibers are immersed in the cryopreservation solution without removing the cells from the hollow fibers. As described above, the hollow fiber hole does not allow the cells to pass through, but allows the culture solution or cryopreservation solution to pass therethrough, so that the inside of the hollow fiber is maintained while maintaining the state in which the cells are encapsulated in the inner space of the hollow fiber. The culture medium in the space or the intracellular solution of the cells can be replaced with a cryopreservation solution. Then, the cells in the hollow fiber are frozen by immersing the hollow fiber in liquid nitrogen or the like.

  In order to preserve the hollow fiber containing the frozen cells, a part of the hollow fiber where the cells are present is cut from the first tube, and the hollow fiber is separated from the first tube. Then, the separated hollow fiber is frozen and stored in a deep freezer or the like. Moreover, without cutting the hollow fiber from the first tubular body, the first tubular body to which the hollow fiber is connected is removed from the drinking device, etc., and the hollow fiber and the first tubular body are made into a container suitable for freezing storage. It may be sealed and stored frozen in a deep freezer.

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

  (2) The core material may protrude from both ends of the hollow fiber.

  Thereby, since the both ends of a hollow fiber are supported by a core material, the breakage of the both ends is prevented.

  (3) A step in which the tubular body is externally fitted to the first end of the hollow fiber, the hollow fiber and the tubular body are connected, and the core material is in contact with the first end of the hollow fiber. It may have a part.

  When the core material is inserted from the first end side of the hollow fiber, the stepped portion of the core material contacts the first end of the hollow fiber, and the insertion limit of the core material is determined. Moreover, since the removal of the core material is limited to the direction in which the core material is pulled out from the first end side, the core material will not fall out of the hollow fiber by its own weight if the first end side of the hollow fiber is vertically upward. .

  (4) In the method for producing a cell device according to the present invention, a core material having a core part that can be inserted into a hollow fiber having both ends opened, and a step part that can come into contact with the first end of the hollow fiber, A first step of inserting the hollow fiber into an insertable tube and positioning the stepped portion at a predetermined position in the tube, and inserting the core of the core into the hollow fiber, A second step of inserting the first end of the yarn into the tube, bringing the first end into contact with the stepped portion of the core, and positioning the hollow fiber relative to the tube; and the hollow fiber A third step of filling an adhesive between the first end side of the tube and the tubular body to bond the hollow fiber and the tubular body.

  In the first step, the core material is inserted to a predetermined position of the tubular body. In the insertion of the core material, the stepped portion of the core material is directed to the side of the tube that is connected to the first end of the hollow fiber. The position where the core material is inserted into the tubular body is determined so that the stepped portion of the core material is disposed at the position where the first end of the hollow fiber is to be positioned in the internal space of the tubular body. The arrangement of the tubular body with the core material may be determined by an appropriate jig or the like.

  In the second step, the first end of the hollow fiber is inserted into the tube while moving the hollow fiber along the core so that the core of the core is inserted through the hollow fiber. The 1st end of the hollow fiber inserted in the pipe body contacts the level | step-difference part of the core material arrange | positioned in the internal space of a pipe body. By this contact, the position for inserting the hollow fiber into the tube body is determined. In this way, the core material is used as a guide for the hollow fiber inserted into the tubular body, and is also used as a jig for positioning the first end of the hollow fiber.

  In the third step, an adhesive is filled between the first end side of the hollow fiber and the tube body. If there is a gap between the hollow fiber and the tube, the gap is filled with an adhesive. If there is almost no gap between the hollow fiber and the tubular body, filling is performed so that an adhesive is applied to the boundary between the hollow fiber and the tubular body. As this adhesive, a so-called medical adhesive is used. Thereby, a tubular body and a hollow fiber are couple | bonded. In the cell device after production, the hollow fiber is supported by the core material, and the hollow fiber is prevented from being bent or damaged. This core material is pulled out from the hollow fiber in use.

  According to the cell device of the present invention, each internal space is continuous as a flow path, and the first tubular body and the hollow fiber are connected. Cells can be taken into the internal space of the thread. The pores of the hollow fiber do not allow cells to pass through, but allow the culture solution or cryopreservation solution to pass through, so that the culture solution in the inner space of the hollow fiber can be maintained while maintaining the state in which the cells are encapsulated in the inner space of the hollow fiber. Or the intracellular solution of the cells can be replaced with a cryopreservation solution.

  Further, since the core material that can be removed during use is inserted into the hollow fiber, the hollow fiber is supported by the core material until use. As a result, the hollow fiber is prevented from being bent due to an external force applied to the hollow fiber coming into contact with another member.

  Moreover, according to the manufacturing method of the device for cells concerning this invention, the core material penetrated by the pipe body functions as a guide of the hollow fiber inserted in a pipe body. Further, since the stepped portion of the core material positioned in the tubular body is in contact with the first end of the hollow fiber inserted into the tubular body, the position at which the hollow fiber is inserted into the tubular body is determined. In addition to functioning as a jig for positioning the first end of the hollow fiber to be inserted into the tubular body, the hollow fiber is supported by the core material after manufacturing, and the hollow fiber is prevented from being bent or damaged.

  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 the overall configuration of a cell device 10 according to an embodiment of the present invention. FIG. 2 is an exploded perspective view of the hollow fiber 11, the injection needle 12, and the core material 13. FIG. 3 is a perspective view showing the overall configuration of the core member 13. 4 to 7 are cross-sectional views for explaining a method for manufacturing the cell device 10. FIG. 8 is a perspective view showing a state where the syringe barrel 15 is connected to the cell device 10 via the tube 14. 9 to 16 are schematic views showing the procedure of the cell cryopreservation method using the cell device 10. 9 to 16, only a part of the hollow fiber 11 and the injection needle 12 of the cell device 10 are shown, and the tube 14 and the syringe barrel 15 are not shown in each drawing.

[Cellular device 10]
As shown in FIGS. 1 and 2, the cell device 10 mainly includes a hollow fiber 11, an injection needle 12, and a core material 13.

  The hollow fiber 11 is formed by forming a membrane having a plurality of pores having a diameter that allows water, electrolyte, protein, and the like to pass therethrough, but does not allow cells to pass, into a straw shape having both ends opened. The inner diameter and length of the hollow fiber 11 are appropriately selected according to the target cells, but the inner diameter is about several hundred μm and the length is about several cm.

  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 polyurethane resin 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. In the present specification, the end of the hollow fiber 11 connected to the injection needle 12 is referred to as a first end 21, and the tip extending from the injection needle 12 is referred to as a second end 22 of the hollow fiber 11. .

  As shown in FIGS. 1 to 3, the core member 13 is a member that is long in the same direction as the axis 101 of the hollow fiber 11 and the injection needle 12, and has a generally elongated cylindrical shape as a whole. The core member 13 has a shape in which a small diameter portion 24 and a large diameter portion 25 are arranged in parallel along the axis 101, and a stepped portion 26 is formed at the boundary between the small diameter portion 24 and the large diameter portion 25.

  The outer diameter of the small diameter portion 24 is substantially the same as or slightly smaller than the inner diameter of the hollow fiber 11. Therefore, the small diameter portion 24 can be inserted into the hollow fiber 11. The length of the small diameter portion 24 along the direction of the axis 101 is slightly longer than the length of the hollow fiber 11. Therefore, in a state where the small diameter portion 24 is inserted through the hollow fiber 11, the small diameter portion 24 can protrude from the first end 21 and the second end 22 that are both ends of the hollow fiber 11. The small diameter portion 24 corresponds to the core portion of the core material in the present invention.

  The outer diameter of the large diameter portion 25 is larger than the inner diameter of the hollow fiber 11 and is substantially the same as or slightly smaller than the inner diameter of the injection needle 12. Therefore, the large diameter portion 25 can be inserted into the injection needle 12 but cannot be inserted into the hollow fiber 11. The length of the large diameter portion 25 along the axis 101 is slightly longer than that of the injection needle 12.

  The step portion 26 formed at the boundary between the small diameter portion 24 and the large diameter portion 25 forms a flat surface extending in a direction orthogonal to the axis 101. The flat surface forming the stepped portion 26 has an annular shape with a dimensional width that is a difference between the outer diameter of the small diameter portion 24 and the outer diameter of the large diameter portion 25. As will be described later, the first end 21 of the hollow fiber 11 can come into contact with the stepped portion 26.

  The core material 13 has a rigidity capable of supporting the hollow fiber 11 so that the hollow fiber 11 is not easily bent in a state where the small diameter portion 24 is inserted into the hollow fiber 11. Although the raw material of the core material 13 is not specifically limited, For example, the core material 13 can be produced from medical stainless steel.

[Method for Manufacturing Cell Device 10]
Below, the manufacturing method of the device 10 for cells mentioned above is demonstrated.

  The manufacturing method of the cell device 10 mainly includes a first step (S1) in which the core material 13 is inserted into the injection needle 12 and the stepped portion 26 is positioned at a predetermined position in the injection needle 12, and the small diameter of the core material 13 The second step of inserting the first end 21 of the hollow fiber 11 into the injection needle 12 so that the portion 24 is inserted through the hollow fiber 11 and positioning the first end 21 in contact with the stepped portion 26 (S2). And a third step (S3) in which the adhesive 27 is filled between the first end 21 side of the hollow fiber 11 and the injection needle 12, and the hollow fiber 11 and the injection needle 12 are coupled.

  As shown in FIG. 4, in the first step (S <b> 1), the core material 13 is inserted to a predetermined position of the injection needle 12. In the insertion of the core material 13, the flat surface of the step portion 26 of the core material 13 faces the side of the injection needle 12 connected to the first end 21 of the hollow fiber 11. That is, the core material 13 is inserted into the injection needle 12 so that the small diameter portion 24 of the core material 13 protrudes from the tip of the injection needle 12.

  As for the position where the core material 13 is inserted into the injection needle 12, the stepped portion 26 of the core material 13 is arranged at the position where the first end 21 of the hollow fiber 11 is to be positioned in the internal space of the injection needle 12. To be determined. Although the jig does not appear in FIG. 4, in the state where the core material 13 is inserted into the injection needle 12, the cannula side of the injection needle 12 and the small diameter portion 24 side of the core material 13 are the upper side, and the hub of the injection needle 12. The injection needle 12 and the core material 13 are erected on a jig (not shown) with the side and the large-diameter portion 25 side of the core material 13 as the lower side. With this jig, the arrangement of the injection needle 12 and the core material 13 is determined, and the step portion 26 of the core material 13 is arranged near the tip of the injection needle 12 as shown in FIG.

  As shown in FIG. 5, in the second step (S <b> 2), the first end 21 of the hollow fiber 11 is inserted into the distal end side of the injection needle 12. When the hollow fiber 11 is slid toward the injection needle 12 along the small diameter portion 24 of the core member 13, the first end 21 of the hollow fiber 11 is inserted into the injection needle 12. That is, the core material 13 is used as a guide for the hollow fiber 11 inserted into the injection needle 12.

  As shown in FIG. 6, the first end 21 of the hollow fiber 11 inserted into the injection needle 12 comes into contact with the step portion 26 of the core member 13 disposed in the internal space of the injection needle 12. By this contact, the position at which the hollow fiber 11 is inserted into the injection needle 12 is determined. That is, the core material 13 is used as a jig for positioning the first end 21 of the hollow fiber 11 inserted into the injection needle 12. As described above, since the step portion 26 of the core member 13 faces upward, the hollow fiber 11 is stationary with the first end 21 supported by the step portion 26.

  As shown in FIG. 7, in the third step (S <b> 3), an adhesive 27 is filled between the first end 21 side of the hollow fiber 11 and the injection needle 12. If there is a gap between the hollow fiber 11 and the injection needle 12, the gap 27 may be filled with an adhesive 27, and the gap between the hollow fiber 11 and the injection needle 12 may be filled. If there is little, it fills so that the adhesive agent 27 may be apply | coated to the boundary with the injection needle 12 along the outer periphery of the hollow fiber 11. As the adhesive 27, a so-called medical adhesive is used. Thereby, the hollow fiber 11 and the injection needle 12 are couple | bonded.

[Cell cryopreservation method]
Hereinafter, a cell cryopreservation method using the cell device 10 will be described.

  The cell cryopreservation method described below mainly includes the first step (S11) of sucking the cells 41 and the culture solution 42 from the cell suspension into the internal space of the hollow fiber 11 and the cells 41 and the culture solution 42. A second step (S12) of immersing the hollow fiber 11 in the vitrification solution 43, a third step (S13) of separating the hollow fiber 11 from the injection needle 12, and a fourth step of cooling the separated hollow fiber 11 ( S14) and four steps.

  When the cell device 10 is used, the core material 13 is removed from the hollow fiber 11. As described above, since the stepped portion 26 of the core material 13 is in contact with the first end 21 of the hollow fiber 11 in the internal space of the injection needle 12, the core material 13 is pulled out from the hub side of the injection needle 12. Thereby, the cell 41 can be sucked into the hollow fiber 11. On the other hand, until the hollow fiber 11 is supported by the core material 13 until it is used, the hollow fiber 11 is prevented from being bent due to the external force applied by the hollow fiber 11 coming into contact with other members. The

  As shown in FIG. 8, the cell device 10 after the core material 13 is removed is connected to the syringe barrel 15 via the tube 14.

  The tube 14 is made of silicone rubber. The inner diameter and outer diameter of the tube 14 are appropriately selected according to the injection needle 12 and the syringe barrel 15. In FIG. 8, the tube 14 is shown with a part omitted in the length direction, but the length of the tube 14 is such that the operator separates the injection needle 12 and the syringe barrel 15 into both hands. The length is suitable for holding and operating, and is usually about several tens of centimeters. The flexibility of the tube 14 is suitable for the suction in which the tube 14 does not buckle and the inner space of the tube 14 is described later when the operator holds the syringe needle 12 and the syringe barrel 15 in both hands. When the negative pressure is increased, the tube 14 is not clogged by closing the internal space. One end of the tube 14 is connected to the hub of the injection needle 12 via a joint or the like. Thereby, the internal space of the injection needle 12 is continuous with the internal space of the tube 14. As this joint or the like, a well-known joint may be used as appropriate, and detailed description thereof is omitted here.

  As long as the tube 14 is flexible, for example, natural rubber, polyurethane, silicone rubber, polyvinyl chloride, polyethylene, polypropylene, an ethylene / propylene copolymer, or the like formed into a tube shape is used as appropriate. It is done. Although the length of the tube 14 is not specifically limited, if the length suitable for operating the injection needle 12 and the syringe barrel 15 separately is adopted and the length is about 50 to 1000 mm, the injection needle 12 is operated with one hand. However, it is preferable that the syringe barrel 15 can be operated with the other hand.

  The configuration of the syringe barrel 15 is common. The syringe 31 has a substantially cylindrical shape, and a distal end side thereof is reduced in diameter to form a cylindrical attachment portion 33. Both ends of the attachment portion 33 are open, and one end communicates with the internal space of the syringe 31. That is, the internal space of the attachment portion 33 forms a port that connects the internal space of the syringe 31 and the outside. The other end of the tube 14 is externally fitted to the attachment portion 33 so that the internal space of the syringe 31 and the internal space of the tube 14 are continuous. The proximal end side of the syringe 31 is opened, and the plunger 32 is inserted into the internal space from the proximal end side. The plunger 32 is longer than the length of the internal space of the syringe 31 in the direction of the axis 101 and is inserted to the innermost part of the internal space of the syringe 31, and a part of the plunger 32 protrudes from the proximal end of the syringe 31 to the outside. Yes.

  Although not shown in each figure, a gasket is connected to the tip of the plunger 32. The gasket is a cylindrical body made of an elastic material such as rubber, and is fitted in the inner space of the syringe 31 in an airtight manner, and can slide along the axis 101 along the inner space of the syringe 31 together with the plunger 32. The direction in which the plunger 32 extends coincides with the sliding direction of the gasket. When the gasket is moved to the proximal end side of the syringe 31, the internal space of the syringe 31 becomes negative pressure, and gas or liquid is sucked from the attachment portion 33 into the internal space.

  The syringe 15 is an example of a suction tool, and other suction tools such as a micropump may be used. However, from the viewpoint of cost and disposal, the disposable syringe 15 is preferable as the suction tool. In addition, the connection between the injection needle 12 and the injection cylinder 15 is not particularly limited, and even if the injection needle 12 is connected directly to the injection cylinder 15 even if connected via the tube 14 as shown in the present embodiment. Good. Further, suction pressure may be applied to the tube 14 from the mouth or the like without using the syringe barrel 15.

  Cells 41 to be cryopreserved are suspended in the culture solution 42. The culture solution 42 is held in a container such as a medium dish. However, in FIGS. 9 to 14, the container is omitted and only a plurality of cells 41 and a part of the culture solution 42 are shown. A plurality of cells 41 are suspended in the culture solution 42, and these cells 41 are of a single type collected from the same individual.

  The culture solution 42 is a liquid suitable for suspending the cells 41, and is selected according to the type and purpose of the cells 41. For example, when culturing sperm cells, oocytes, or early embryos, the TCM-199 medium, Examples thereof include RPMI-1640 medium, DMEM medium, MEM medium, α-MEM medium, and IMDM medium. Moreover, HUCUM culture medium (made by a Nipro company) is mentioned as a culture medium aiming at human in vitro fertilization. Examples of the medium for in vitro fertilization of bovine 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 for in vitro fertilization of pigs include NCSU media and PZM-5 media (manufactured by Functional Peptide Institute).

  As shown in FIG. 9, in the first step (S11), the injection needle 12 of the cell device 10 is operated with one hand, and the second end 22 of the hollow fiber 11 is inserted into the culture solution. Then, the syringe cylinder 15 is operated with the other hand, the plunger 32 is pulled out from the syringe 31, and a small amount of the culture solution 42 is sucked into the hollow fiber 11.

  Subsequently, as shown in FIG. 10, the injection needle 12 is operated to take out the second end 22 of the hollow fiber 11 from the culture solution 42, and the injection cylinder 15 is operated again to allow a small amount of air to be contained inside the hollow fiber 11. Aspirate into space. Thereby, the trace amount culture solution 42 sucked into the hollow fiber 11 slightly moves in the inner space of the hollow fiber 11 to the injection needle 12 side.

  Next, as shown in FIG. 11, the injection needle 12 is operated to insert the second end 22 of the hollow fiber 11 into the culture solution 42 again, and the cells 41 in the culture solution 42 are confirmed using a microscope. However, the second end 22 of the hollow fiber 11 is brought close to the cell 41. Then, the syringe cylinder 15 is operated to suck the cells 41 together with the culture solution 42 into the internal space of the hollow fiber 11. Thereby, in the internal space of the hollow fiber 11, a trace gas layer 45 is formed on the first end 21 side from the liquid layer 44 in which the cells 41 and the culture solution 42 exist. That is, in the inner space of the hollow fiber 11, a layer including only the culture solution 42, a gas layer 45, a cell 41, and a liquid layer 44 including the culture solution 42 are sequentially provided from the first end 21 side to the second end 22 side. Each is formed.

  Next, as shown in FIG. 12, the second end 22 of the hollow fiber 11 is not taken out from the culture solution 42, the second end 22 is brought close to another cell 41, the syringe cylinder 15 is operated, and the hollow fiber 11 is hollow. Other cells 41 are sucked into the internal space of the thread 11 together with the culture solution 42. The number of cells 41 to be sucked into the inner space of the hollow fiber 11 is not particularly limited, but in this embodiment, three cells 41 are sucked into the inner space of the hollow fiber 11 together with the culture solution 42 to form a liquid layer 44. ing.

  After the three cells 41 are sucked into the internal space of the hollow fiber 11, the syringe needle 12 is operated to take out the second end 22 of the hollow fiber 11 from the culture solution 42 as shown in FIG. To suck a small amount of air into the internal space of the hollow fiber 11. Thereby, the liquid layer 44 and the gas layer 45 in the hollow fiber 11 slightly move in the inner space of the hollow fiber 11 to the injection needle 12 side.

  Next, as shown in FIG. 14, the injection needle 12 is operated to insert the second end 22 of the hollow fiber 11 into the culture solution 42 again, and the injection cylinder 15 is operated to change the internal space of the hollow fiber 11. Aspirate the culture solution 42. Thereby, a small amount of gas layer 46 is formed on the second end 22 side with respect to the liquid layer 44.

  In the second step (S 12), as shown in FIG. 15, the hollow fiber 11 containing the cells 41 and the culture solution 42 is immersed in the vitrification solution 43 in the internal space. As described above, the hole of the hollow fiber 11 does not allow the cell 41 to pass through, but allows the culture solution 42 and the vitrification solution 43 to pass therethrough, so that the state where the cell 41 is included in the internal space of the hollow fiber 11 is maintained. However, the vitrification solution 43 replaces the culture solution 42 in the internal space of the hollow fiber 11 and the intracellular solution of the cells 41.

  In addition, when replacing the culture solution 42 in the hollow fiber 11 with the vitrification solution 43, when it is desired to select a slow condition for the cells 41, the culture solution 42 has an appropriate concentration gradient as the vitrification solution 43. A plurality of kinds mixed in the above are used. Such concentration gradients are set to 2 to 5 types, for example. When a plurality of types of vitrification solutions 43 are used, preliminarily prepared vitrification solutions 43 are prepared, and the hollow fibers 11 are added to the plurality of types of vitrification solutions 43 as shown in FIG. Soaked sequentially.

  Examples of the cryopreservation method for cells include quick freezing in a high-concentration freezing protection liquid and normal freezing in a low-concentration freezing protection liquid. Rapid freezing in a high concentration frost damage protection liquid is also referred to as vitrification, and the frost damage protection 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.

  As shown in FIG. 16, in the third step (S <b> 13), the hollow fiber 11 containing the cell 41 is separated from the injection needle 12. This separation is performed by cutting the hollow fiber 11 with a scissors or the like on the first end 21 side from the liquid layer 44 and the gas layer 45 containing the cells 41. As a result, the hollow fiber 11 containing the cells 41 and the vitrification solution 43 is separated from the cell device 10.

  Although not shown in the figure, in the fourth step (S14), the hollow fiber 11 separated from the cell device 10 is put into liquid nitrogen or liquid helium and cooled. Thereby, the cell 41 included in the internal space of the hollow fiber 11 is vitrified. The hollow fiber 11 containing the vitrified cells 41 is put into a suitable container and stored in a freezer.

  As described above, the cells 41 encapsulated 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. After the thawing is completed, when a discharge pressure is applied from the first end 21 or the second end 22 of the hollow fiber 11 to the internal space, the cells 41 are discharged from the internal space of the hollow fiber 11 to the outside. Further, if culturing is performed in a state where the cells 41 are encapsulated in the internal space of the hollow fiber 11, the hollow fiber 11 encapsulating the cells 41 may be put into a desired medium after thawing.

[Operational effects of this embodiment]
According to the cell device 10 and the cell cryopreservation method according to the present embodiment, the hollow spaces 11 and the injection needles 12 are connected to each other through the internal spaces as the flow paths. When a suction pressure is applied to the cells, the cells 41 can be taken into the internal space of the hollow fiber 11. The holes of the hollow fiber 11 do not allow the cells 41 to pass through, but allow the culture solution 42 and the vitrification solution 43 to pass therethrough. The vitrification solution 43 can replace the culture solution 42 in the internal space 11 and the intracellular solution of the cells 41.

  Moreover, since the core material 13 that can be removed during use is inserted through the hollow fiber 11, the hollow fiber 11 is supported by the core material 13 until use. As a result, the hollow fiber 11 is prevented from being bent due to the external force being applied to the other member or the hollow fiber 11 being bent.

  Further, since the core material 13 protrudes from the first end 21 and the second end 22 which are both ends of the hollow fiber 11, both ends of the hollow fiber 11 are supported by the core material 13, and the both ends of the hollow fiber 11 are damaged. Is prevented.

  Moreover, since the core material 13 has the step part 26 which contact | abuts to the 1st end 21 of the hollow fiber 11, if the core material 13 is inserted from the 1st end 21 side of the hollow fiber 11, the level difference part 36 of the core material 13 will become. The insertion limit of the core material 13 is determined by contacting the first end 21 of the hollow fiber 11. Further, the removal of the core material 13 is limited to the direction of pulling out from the first end 21 side. Therefore, if the first end 21 side of the hollow fiber 11 is directed vertically upward, the core material 13 is pulled by the own weight. Will not fall off.

  Further, according to the method for manufacturing the cell device 10 according to this embodiment, the small diameter portion 24 of the core member 13 inserted through the injection needle 12 functions as a guide for the hollow fiber 11 inserted into the injection needle 12. Further, the stepped portion 36 of the core member 13 positioned in the injection needle 12 is in contact with the first end 21 of the hollow fiber 11 inserted into the injection needle 12, and the position where the hollow fiber 11 is inserted into the injection needle 12. Therefore, the core material 13 functions as a jig for positioning the first end 21 of the hollow fiber 11 inserted into the injection needle 12, and the hollow fiber 11 is supported by the core material 13 after manufacture. Thus, the hollow fiber 11 is prevented from being bent or damaged.

FIG. 1 is a perspective view showing the overall configuration of a cell device 10 according to an embodiment of the present invention. FIG. 2 is an exploded perspective view of the hollow fiber 11, the injection needle 12, and the core material 13. FIG. 3 is a perspective view showing the overall configuration of the core member 13. FIG. 4 is a cross-sectional view for explaining the method for manufacturing the cell device 10. FIG. 5 is a cross-sectional view for explaining the method for manufacturing the cell device 10. FIG. 6 is a cross-sectional view for explaining the method for manufacturing the cell device 10. FIG. 7 is a cross-sectional view for explaining the method for manufacturing the cell device 10. FIG. 8 is a perspective view showing a state where the syringe barrel 15 is connected to the cell device 10 via the tube 14. FIG. 9 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10. FIG. 10 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10. FIG. 11 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10. FIG. 12 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10. FIG. 13 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10. FIG. 14 is a schematic diagram showing a procedure of a cell cryopreservation method using the cell device 10. FIG. 15 is a schematic diagram showing a procedure of a cell cryopreservation method using the cell device 10. FIG. 16 is a schematic diagram showing the procedure of a cell cryopreservation method using the cell device 10.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Cell device 11 ... Hollow fiber 12 ... Injection needle (tube body)
13 ... Core 21 ... First end 24 ... Small diameter part (core part)
26 ... Step part

Claims (4)

Hollow fibers open at both ends;
A tubular body connected to the first end of the hollow fiber and constituting a flow path whose internal space is continuous with the internal space of the hollow fiber;
A core material inserted through the hollow fiber,
The device for cells, wherein the core material is removable from the hollow fiber in use.   The cell device according to claim 1, wherein the core material is protruded from both ends of the hollow fiber. The tubular body is externally fitted to the first end of the hollow fiber, the hollow fiber and the tubular body are connected,
The device for a cell according to claim 1 or 2, wherein the core material has a stepped portion that comes into contact with the first end of the hollow fiber. Inserting a core material having a core part that can be inserted into a hollow fiber having both ends open and a step part that can come into contact with the first end of the hollow fiber into a tubular body into which the hollow fiber can be inserted, A first step of positioning the stepped portion at a predetermined position in the tube;
The core portion of the core material is inserted into the hollow fiber, the first end of the hollow fiber is inserted into the tube body, the first end is brought into contact with the step portion of the core material, and the tube A second step of positioning the hollow fiber relative to the body;
A method for producing a device for a cell, comprising: a third step of filling an adhesive between the first end side of the hollow fiber and the tubular body to bond the hollow fiber and the tubular body.

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