WO2015146404A1 - Platelet production device and platelet production method - Google Patents

Platelet production device and platelet production method Download PDF

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Publication number
WO2015146404A1
WO2015146404A1 PCT/JP2015/054915 JP2015054915W WO2015146404A1 WO 2015146404 A1 WO2015146404 A1 WO 2015146404A1 JP 2015054915 W JP2015054915 W JP 2015054915W WO 2015146404 A1 WO2015146404 A1 WO 2015146404A1
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WO
WIPO (PCT)
Prior art keywords
platelet production
megakaryocytes
platelets
holding member
megakaryocyte
Prior art date
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PCT/JP2015/054915
Other languages
French (fr)
Japanese (ja)
Inventor
雄一郎 津田
元井　昌司
豊治 寺田
義生 野上
和正 柴田
Original Assignee
東レエンジニアリング株式会社
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Application filed by 東レエンジニアリング株式会社 filed Critical 東レエンジニアリング株式会社
Publication of WO2015146404A1 publication Critical patent/WO2015146404A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus

Definitions

  • the present invention relates to a platelet production apparatus and a platelet production method. More specifically, the present invention relates to an apparatus and method for producing platelets from megakaryocytes derived from iPS cells.
  • platelets are separated from blood by a centrifugal separation method using a difference in specific gravity.
  • a centrifugal separation method using a difference in specific gravity.
  • the centrifugal separator described in Patent Document 1 is provided for each specific gravity of each blood cell in the blood storage space formed at the outer edge of the centrifugal bowl by rotating the centrifugal bowl of the centrifuge into which blood has flowed at high speed. , Separated into plasma layer, buffy coat layer and red blood cell layer. Then, the platelets are separated from the buffy coat layer by centrifugation.
  • megakaryocytes which are nucleated cells remaining after platelet production without producing platelets.
  • specific gravity of megakaryocytes and platelets is close, in order to separate megakaryocytes and platelets by centrifugation, it is necessary to add a large centrifugal acceleration. For this reason, even if megakaryocytes and platelets can be separated by the centrifugal separation method, there is a problem that the ratio of platelets destroyed by a large centrifugal acceleration increases and the platelet recovery rate decreases.
  • An object of the present invention is to provide a platelet production apparatus and a platelet production method capable of efficiently producing platelets.
  • the present invention is a container for storing a liquid containing megakaryocytes, a supply means for supplying a liquid containing megakaryocytes in the container, a holding means provided in a part of the container and holding the megakaryocytes, An external force applying means for applying an external force to the megakaryocyte held by the holding means to produce platelets.
  • the holding means is formed with an opening having a size that allows the platelets to pass therethrough and the megakaryocytes cannot pass through, and a megakaryocyte is held in one part of the opening by holding the megakaryocyte. It is.
  • the external force applying means applies an external force to the megakaryocyte by a fluid flow from the other side of the opening.
  • the external force applying means applies an external force to the megakaryocyte by creating a pressure difference between one side and the other side of the opening.
  • the external force applying means applies an external force to the megakaryocyte by generating a charge on the other side of the opening that is different from the charge charged on the megakaryocyte.
  • the present invention holds megakaryocytes and applies force to the megakaryocytes from the outside to produce platelets from the megakaryocytes.
  • the step of separating megakaryocytes and platelets is omitted. Thereby, platelets can be produced efficiently.
  • the force applied to the megakaryocyte is set to an arbitrary value, and platelets are produced from the other side of the opening of the holding means. Thereby, platelets can be produced efficiently.
  • each member constituting the platelet production device is made of an appropriate material from the application and strength among non-reactive polymer, biocompatible metal, glass and the like unless otherwise specified.
  • the non-reactive polymer include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, halogenated polymers such as polyvinyl chloride, polyamide, polyimide polycarbonate, polyethylene, polypropylene, and polystyrene.
  • the biocompatible metal include stainless steel, titanium, platinum and alloys thereof, and cobalt chromium alloy.
  • the platelet production apparatus 1 produces and collects platelets from megakaryocytes.
  • the platelet production apparatus 1 is configured as a double circulation system that produces platelets while circulating a liquid containing megakaryocytes and a liquid containing platelets together.
  • the platelet production apparatus 1 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 6, a collection channel 7, a collection pump 8, and a collection tank 9.
  • the storage tank 2 holds a liquid (hereinafter simply referred to as “culture medium”) containing mature megakaryocytes and platelets produced from a part of the mature megakaryocytes.
  • the storage tank 2 is configured to be able to supply a culture solution from the outside.
  • the storage tank 2 is connected to a culture device 23 and the like which will be described later, and is configured to be supplied with a culture solution produced by the culture device 23 and the like.
  • the circulation channel 3 is a channel for circulating the culture solution.
  • the circulation channel 3 is composed of a tube made of a non-reactive polymer, a biocompatible metal, glass or the like.
  • the circulation channel 3 is configured to connect the storage tank 2 and the platelet production chamber 5 via the supply pump 4.
  • the circulation channel 3 is configured to connect the platelet production chamber 5 and the storage tank 2. That is, the culture solution in the storage tank 2 is configured to be supplied to the platelet production chamber 5 through the circulation channel 3 by the supply pump 4. Then, the culture solution supplied to the platelet production chamber 5 is configured to be returned to the storage tank 2 again through the circulation channel 3.
  • the supply pump 4 serving as a supply means supplies the culture solution in the storage tank 2 to the circulation channel 3.
  • the supply pump 4 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this.
  • the supply pump 4 is provided in the middle of the circulation channel 3.
  • the supply pump 4 is configured to circulate the culture solution between the storage tank 2 and the platelet production chamber 5 by supplying the culture solution in the storage tank 2 to the circulation channel 3.
  • the supply pump 4 is configured so that the culture solution can be supplied to the circulation channel 3 at an arbitrary flow rate.
  • the platelet production device 1 is configured to supply the culture solution by the supply pump 4, but is not limited thereto.
  • the platelet production chamber 5 which is a container, constitutes a space for producing platelets from megakaryocytes.
  • the platelet production chamber 5 is a container formed from a non-reactive polymer or a biocompatible metal.
  • the platelet production chamber 5 is provided in the middle of the circulation channel 3. That is, the platelet production chamber 5 is configured so that the culture solution is supplied from the storage tank 2 through the circulation channel 3 and the culture solution in the platelet production chamber 5 is returned to the storage tank 2.
  • the holding member 6 which is a holding means holds a megakaryocyte.
  • the holding member 6 is formed with a plurality of openings 6a so as to communicate from one side surface to the other side surface of a thin plate (or filter) made of a non-reactive polymer or a biocompatible metal (see FIG. 2).
  • the holding member 6 is provided in the platelet production chamber 5 so as to partition a part of the space. That is, one side surface of the holding member 6 is arranged inside the platelet production chamber 5 so that megakaryocytes contained in the culture solution are in contact with the opening 6a.
  • the other side surface of the holding member 6 constitutes a part of the collection flow path 7 in the platelet production chamber 5.
  • the holding member 6 is configured by forming a thin plate or the like into a hollow tubular shape.
  • the holding member 6 has an opening 6a formed so as to communicate the outer side of the tube that is one side surface and the inner side of the tube that is the other side surface.
  • the opening 6a has a diameter D3 in the case of the hole shown in FIG. 2B or a slit width D2 in the case of the slit shown in FIG.
  • the size of megakaryocytes derived from iPS cells varies depending on the culture process. Therefore, it is desirable to determine the hole diameter D1 and the slit width D2 based on the size of the megakaryocyte at that time.
  • the holding member 6 is disposed in the platelet production chamber 5, and a recovery channel 7 is connected to both ends thereof.
  • the cross-sectional shape of the holding member 6 is not limited to a circular shape, and may be a rectangular or elliptical cross-sectional shape, for example.
  • the size and number of the holding members 6 provided in the platelet production chamber 5 are not particularly limited as long as they do not hinder the flow of the culture solution in the platelet production chamber 5.
  • the surface area of the holding member 6 is increased by increasing the size of the tube of the holding member 6 as much as possible or providing a plurality of holding members 6 in the platelet production chamber 5. May be increased.
  • the recovery channel 7 is a liquid for recovering platelets contained in the culture medium and platelets already produced from megakaryocytes (hereinafter simply referred to as “recovery liquid”). It is a flow path through which.
  • the collection channel 7 is configured to connect one end (inlet side) end of the holding member 6 and the collection tank 9.
  • the recovery channel 7 is configured to connect the other end (exit side) end of the holding member 6 and the recovery tank 9.
  • the recovery pump 8 which is an external force applying means supplies the recovery liquid in the recovery tank 9 to the recovery flow path 7.
  • the supply pump 4 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this.
  • the recovery pump 8 is provided in the middle of the recovery flow path 7 that connects one end (inlet side) of the holding member 6 and the recovery tank 9.
  • the recovery pump 8 is configured to circulate the recovery liquid between the holding member 6 and the recovery tank 9 by supplying the recovery liquid in the recovery tank 9 to the recovery flow path 7.
  • the recovery pump 8 is configured to supply the recovery liquid to the recovery flow path 7 at an arbitrary flow rate.
  • the recovery liquid is supplied by the recovery pump 8, but the present invention is not limited to this.
  • the double-circulation type platelet production apparatus 1 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 5 through the circulation channel 3 by the supply pump 4 (FIG. 2). (See a thick arrow). Further, the platelet production device 1 is configured such that the culture solution supplied to the platelet production chamber 5 is discharged to the storage tank 2 through the circulation channel 3. That is, the platelet production apparatus 1 is configured so that the culture solution circulates between the storage tank 2 and the platelet production chamber 5. The platelet production apparatus 1 is configured such that the collected liquid in the collection tank 9 is supplied to the inside of the holding member 6 through the collection channel 7 by the collection pump 8.
  • the platelet production device 1 is configured such that the collected liquid supplied into the holding member 6 is discharged to the collection tank 9 through the collection channel 7. That is, the platelet production apparatus 1 is configured such that the collected liquid circulates between the collection tank 9 and the holding member 6 (see the white arrow in FIG. 2A).
  • the embodiment of the platelet production method in the platelet production apparatus 1 will be specifically described with reference to FIGS.
  • the description will be made assuming that the opening 6a of the holding member 6 is formed in a hole shape.
  • the platelet production apparatus 1 supplies the culture solution in the storage tank 2 to the circulation channel 3 by the supply pump 4. Thereby, the megakaryocytes contained in the culture solution and the platelets already produced from the megakaryocytes are supplied to the platelet production chamber 5 through the circulation channel 3.
  • some megakaryocytes A of the megakaryocytes supplied to the platelet production chamber 5 together with the culture solution come into contact with the holding member 6.
  • the megakaryocyte A in contact with the holding member 6 is held by the holding member 6 with a part of the megakaryocyte A fitted into one side of the opening 6 a provided in the holding member 6. Since the particle size of the megakaryocyte is larger than the diameter D1 (see FIG. 2B) of the opening 6a, it cannot pass through the opening 6a.
  • some of the platelets supplied to the platelet production chamber 5 together with the culture solution enter the opening 6 a of the holding member 6.
  • the particle size of platelets is smaller than the diameter D1 of the opening 6a, it can pass through the opening 6a. That is, only some platelets enter the inside of the holding member 6 from the opening 6a out of some megakaryocytes A and platelets in contact with the holding member 6.
  • megakaryocytes contained in the culture solution megakaryocytes that did not contact the holding member 6 and megakaryocytes B that contacted the holding member 6 but did not get stuck in the opening 6a were discharged from the platelet production chamber 5 together with the culture solution. And returned to the storage tank 2.
  • the platelets contained in the culture solution the platelets that have passed through the opening 6a and have not entered the holding member 6 are discharged together with the culture solution from the platelet production chamber 5 and returned to the storage tank 2.
  • the megakaryocytes and platelets returned to the storage tank 2 are supplied again to the platelet production chamber 5 through the circulation channel 3 together with other megakaryocytes and platelets by the supply pump 4.
  • the megakaryocyte A held by the holding member 6 is circulated in the recovery flow path 7 by a recovery pump 8 which is an external force applying means in a portion that is fitted in the opening 6a.
  • the pushing force is applied from the collected liquid in the flow direction (see thin arrows in FIG. 3).
  • the platelet production apparatus 1 can control the force applied to the megakaryocyte A by controlling the flow rate of the collection pump 8. In the megakaryocyte A pushed in the flow direction of the collected liquid, a shear stress is generated at a position where a force due to the flow of the collected liquid is applied and a contact portion with the opening 6a.
  • a part of the cytoplasm forming the megakaryocyte A is sheared starting from a location where shear stress is generated by the flow of the recovered liquid. That is, in the megakaryocyte A, a part of its cytoplasm is separated by the force caused by the flow of the recovered liquid, and platelets C are produced. The produced platelets are mixed in the recovery liquid and conveyed to the recovery tank 9 through the recovery flow path 7 together with the recovery liquid.
  • the megakaryocytes held by the holding member 6 continue to produce platelets while the force due to the flow of the collected liquid is applied.
  • Megakaryocytes separated from the holding member 6 stop producing platelets, are discharged from the platelet production chamber 5 together with the culture solution, and are returned to the storage tank 2. Further, megakaryocytes held again by the other holding member 6 in the platelet production chamber 5 resume production of platelets.
  • Megakaryocytes and platelets discharged from the platelet production chamber 5 and returned to the storage tank 2 are supplied again to the platelet production chamber 5 through the circulation channel 3 together with other megakaryocytes and platelets by the supply pump 4.
  • the platelet production device 1 produces platelets by applying force due to the flow of the collected liquid to the megakaryocytes held by the holding member 6. Furthermore, since the platelet production apparatus 1 collects only the produced platelets and collects them in the collection solution, the step of separating megakaryocytes and platelets is omitted. Moreover, the platelet production apparatus 1 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the collection pump 8. Thereby, the platelet production apparatus 1 can produce platelets efficiently.
  • the platelet production apparatus 10 in the second embodiment of the platelet production apparatus according to the present invention will be described with reference to FIGS. 4 and 5.
  • the same points as those of the above-described embodiments will not be specifically described, and different portions will be mainly described.
  • the platelet production apparatus 10 is configured as a circulating suction system that produces platelets by suction (pressure difference) while circulating a liquid containing megakaryocytes.
  • the platelet production apparatus 10 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 11, a collection channel 7, a suction pump 12, and a collection tank 9.
  • the configuration for circulating the culture solution of the platelet production device 10 is the same as the configuration of the platelet production device 1 according to the first embodiment described above.
  • the holding member 11 as the holding means is formed in a hollow tubular shape with one end closed.
  • the holding member 11 has an opening portion 11a formed so as to communicate the outside of the tube that is one side surface and the inside of the tube that is the other side surface.
  • the holding member 11 is disposed in the platelet production chamber 5.
  • the other end of the holding member 11 is connected to the recovery tank 9 via the recovery flow path 7.
  • the size and number of the holding members 11 provided in the storage tank 2 are not particularly limited.
  • the surface area of the holding member 11 is increased by increasing the size of the tube of the holding member 11 as much as possible or by providing a plurality of holding members 11 in the platelet production chamber 5. May be increased.
  • the suction pump 12 which is an external force applying means, makes the pressure in the recovery channel 7 lower than the pressure in the circulation channel 3.
  • the suction pump 12 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this.
  • the suction pump 12 is provided in the middle of the recovery flow path 7 that connects the holding member 11 and the recovery tank 9. Thereby, the suction pump 12 can reduce the pressure inside the holding member 11 connected to the recovery flow path 7 by sucking the inside of the recovery flow path 7 (FIG. 5A) See arrow). That is, the platelet production apparatus 10 is configured to generate a pressure difference between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 5 that is outside the holding member 11.
  • the suction pump 12 can set the pressure of the recovery flow path 7 to an arbitrary pressure by changing the flow rate.
  • the circulatory suction type platelet production apparatus 10 configured as described above is configured so that the culture medium circulates between the storage tank 2 and the platelet production chamber 5.
  • the platelet production apparatus 10 is configured such that a pressure difference is generated between the pressure inside the holding member 11 and the pressure in the platelet production chamber 5 outside the holding member 11 by the suction pump 12.
  • megakaryocytes supplied to the platelet production chamber 5 together with the culture solution and platelets already produced from the megakaryocytes are retained by some megakaryocytes and platelets.
  • Part of the opening 11 a provided in the member 11 is fitted into one side and held by the holding member 11.
  • the holding member 11 has an opening 11a in the platelet production chamber 5 due to a pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the recovery flow path 7.
  • a force for sucking the culture solution into the collection channel 7 is generated (see thin arrows).
  • Megakaryocytes and platelets within the range of the suction force generated in the opening 11a are sucked toward the opening 11a.
  • a part of the megakaryocytes A sucked into the opening portion 11a is fitted into one side of the opening portion 11a and held by the holding member 11.
  • the megakaryocyte A held by the holding member 11 has a force in a direction in which it is drawn into the collection channel 7 due to a pressure difference between the platelet production chamber 5 and the collection channel 7 generated by the suction pump 12 which is an external force applying means. Will be added. That is, the megakaryocyte is pressed against the holding member 11 by a force drawn into the holding member 11. At this time, only the force (suction force) due to the pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the collection channel 7 is applied to the megakaryocyte A. Therefore, the platelet production apparatus 10 can control the force applied to the megakaryocyte by controlling the flow rate of the suction pump 12.
  • the platelet production device 10 applies a force due to a pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the collection channel 7 to the megakaryocyte held by the holding member 11. To produce platelets. Furthermore, since only the produced platelets are mixed in the collected liquid through the opening 11a of the holding member 11, the platelet producing apparatus 10 omits the step of separating megakaryocytes and platelets. Moreover, the platelet production apparatus 10 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the suction pump 12. Thereby, the platelet production apparatus 10 can produce platelets efficiently.
  • the platelet production device 13 is configured as a circulating charge system that produces platelets by Coulomb force generated between charges while circulating a liquid containing megakaryocytes.
  • the platelet production device 13 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 14, a collection tank 15, and a charge generation device 16.
  • the configuration for circulating the culture solution of the platelet production device 13 is the same as the configuration of the platelet production device 1 according to the first embodiment described above.
  • the holding member 14 as a holding means is formed in a plate shape.
  • the holding member 14 has an opening 14a so as to communicate one side and the other side.
  • the holding member 14 is provided in the platelet production chamber 5 so as to partition the space. That is, one side surface of the holding member 14 is configured so that the megakaryocyte disposed in the platelet production chamber 5 and contained in the culture solution contacts the opening 14a (one side) of the holding member 14. .
  • the other side surface of the holding member 14 constitutes a part of the collection flow path 7 in the platelet production chamber 5.
  • the holding member 14 is disposed at the bottom of the platelet production chamber 5, and one side surface thereof constitutes the bottom surface of the platelet production chamber 5.
  • the installation mode of the holding member 14 provided in the platelet production chamber 5 is not particularly limited.
  • the platelet production chamber 5 may be arranged in the middle of the left-right direction of the platelet production chamber 5 and the platelet production chamber 5 may be divided into left and right.
  • the platelet production chamber 5 may be divided into a plurality of spaces by the plurality of holding members 14 to increase the surface area of the holding member 14. .
  • the collection tank 15 collects the produced platelets.
  • the collection tank 15 is disposed below the platelet production chamber 5 and connected so as to cover the bottom of the platelet production chamber 5. That is, the upper surface of the collection tank 15 is configured from the other side surface of the holding member 14. Thereby, the collection tank 15 is connected to the platelet production chamber 5 via the holding member 14. Further, the collection tank 15 is communicated with the platelet production chamber 5 via the opening 14 a of the holding member 14.
  • the charge generator 16 which is an external force applying means generates positive charges.
  • the charge generation device 16 is configured such that the electrode 16 a is disposed in the vicinity of the holding member 14 in the collection tank 15 and generates a positive charge in the vicinity of the other side surface of the holding member 14.
  • the charge generator 16 is configured to generate an arbitrary amount of positive charges.
  • the thus configured circulating charge type platelet production apparatus 13 is configured such that the culture medium circulates between the storage tank 2 and the platelet production chamber 5.
  • the platelet production device 13 is configured such that a Coulomb force is generated between the platelet generator 13 and the megakaryocyte charged with a negative charge by generating a positive charge with the charge generator 16.
  • the megakaryocytes supplied to the platelet production chamber 5 together with the culture solution and the platelets already produced from the megakaryocytes are provided in the holding member 14 with some megakaryocytes A and platelets.
  • a portion of the opening 14 a is fitted into one side of the opening 14 a and is held by the holding member 14.
  • some of the platelets supplied to the platelet production chamber 5 together with the culture solution enter the opening 14 a of the holding member 14.
  • Megakaryocytes are always negatively charged.
  • the megakaryocyte A held by the holding member 14 is placed in the recovery tank 15 by the Coulomb force generated between the positive charge generated in the recovery tank 15 by the charge generator 16 which is an external force applying means.
  • a pulling force is applied (see thin arrows). That is, the megakaryocyte A is pressed against the holding member 14 by the force drawn into the collection tank 15. At this time, only the force in the direction of being drawn into the recovery tank 15 by the Coulomb force is applied to the megakaryocyte. Therefore, the platelet production device 13 can control the force applied to the megakaryocyte by controlling the charge generation amount of the charge generation device 16.
  • a shear stress is generated at a portion where a Coulomb force is applied or a contact portion with the opening 14 a of the holding member 14.
  • the part of the cytoplasm forming the megakaryocyte is sheared from the location where the shear stress is generated by the Coulomb force to the megakaryocyte drawn into the recovery tank 15.
  • a part of the cytoplasm is separated by Coulomb force and platelets C are produced.
  • the produced platelet C is collected in a state separated from megakaryocytes by being mixed in the collection liquid in the collection tank 15.
  • the platelet production device 13 produces platelets by applying a Coulomb force in the direction of being drawn into the collection tank 15 to the megakaryocytes held by the holding member 14. Furthermore, since only the produced platelets are collected through the opening 14a of the holding member 14, the platelet producing device 13 omits the step of separating megakaryocytes and platelets. In addition, the platelet production device 13 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the charge generation amount of the charge generation device 16. Thereby, the platelet production apparatus 13 can produce platelets efficiently.
  • the platelet production device 17 is configured as an agitation circulation system that produces platelets by circulating the collected liquid while expanding the liquid containing megakaryocytes.
  • the platelet production apparatus 17 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 6, a collection flow path 7, a collection pump 8, and a collection tank 9.
  • the supply channel 18 is a channel for supplying a culture solution.
  • the supply flow path 18 is comprised from piping which consists of a non-reactive polymer, a bioaffinity metal, glass, etc.
  • the supply channel 18 is configured to connect the storage tank 2 and the platelet production chamber 19 via the supply pump 4. That is, the culture solution in the storage tank 2 is configured to be supplied to the platelet production chamber 19 through the supply channel 18 by the supply pump 4 provided in the middle of the supply channel 18.
  • the platelet production chamber 19 constitutes a space for producing platelets from megakaryocytes.
  • the platelet production chamber 19 is a container formed from a non-reactive polymer or a biocompatible metal.
  • the platelet production chamber 19 is connected to the storage tank 2 through the supply channel 18. That is, the platelet production chamber 19 is configured so that the culture solution is supplied from the storage tank 2 through the supply channel 18.
  • the platelet production chamber 19 is provided with a stirring device 19a for stirring the supplied culture solution.
  • the agitation and circulation type platelet production apparatus 17 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Further, the platelet production device 17 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. Further, the platelet production device 17 is configured such that the collected liquid in the collection tank 9 is supplied into the holding member 6 through the collection channel 7 by the collection pump 8. Further, the platelet production device 1 is configured such that the collected liquid supplied into the holding member 6 is discharged to the collection tank 9 through the collection channel 7. That is, the platelet production device 17 is configured such that the collected liquid circulates between the collection tank 9 and the holding member 6.
  • the megakaryocytes supplied to the platelet production chamber 19 together with the culture solution and the platelets already produced from the megakaryocytes are platelets.
  • some megakaryocytes and platelets contact the holding member 6.
  • a part of the megakaryocytes in contact with the holding member 6 is held by the holding member 6 while part of the megakaryocyte is fitted into one side of the opening 6 a provided in the holding member 6.
  • the held megakaryocyte is subjected to a force due to the flow of the recovery liquid supplied by the recovery pump 8.
  • Megakaryocytes In the megakaryocyte pushed in the flow direction of the collected liquid, a shear stress is generated at a portion where a force due to the flow of the collected liquid is applied or a contact portion with the opening 6 a of the holding member 6. Megakaryocytes produce platelets by separating a part of their cytoplasm by the force of the flow of the collected liquid.
  • megakaryocytes contained in the culture solution were stirred by the stirring device 19 a of the platelet production chamber 19. It is moved in the direction in contact with the holding member 6 by the flow of the culture broth (see thin arrows).
  • the platelet production device 17 produces platelets by applying a force due to the flow of the recovered liquid to the megakaryocytes held by the holding member 6. Furthermore, since the platelet production apparatus 17 collects only the produced platelets and collects them in the collection liquid, the step of separating megakaryocytes and platelets is omitted. Further, the platelet production device 17 is set so that the force applied to the megakaryocyte becomes a value appropriate for the production of platelets by changing the flow rate of the collection pump 8. Thereby, the platelet production apparatus 17 can produce platelets efficiently.
  • the platelet production device 20 has a configuration in which the culture solution of the platelet production device 10 according to the second embodiment described above is circulated by the supply pump 4 and is agitated by the agitator 19 a, and suction (pressure difference) ) Is configured as a stirring and aspiration method for producing platelets.
  • the stirring suction type platelet production apparatus 20 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 11, a collection flow path 7, a suction pump 12, and a collection tank 9.
  • the configuration of stirring the culture solution in the platelet production chamber 19 of the platelet production device 20 is the same as the configuration of the platelet production device 17 in the fourth embodiment described above.
  • the configuration for applying force by suction (pressure difference) to the megakaryocyte held by the holding member 11 of the platelet production device 20 is the same as the configuration of the platelet production device 10 in the second embodiment described above.
  • the agitation and suction type platelet production apparatus 20 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Further, the platelet production device 20 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. In addition, the platelet production device 20 is configured such that a pressure difference is generated between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 19 that is outside the holding member 11 by the suction pump 12.
  • the platelet production apparatus 20 produces platelets by applying a force due to a pressure difference between the platelet production chamber 19 and the collection flow path 7 to the megakaryocytes held by the holding member 11. Furthermore, since only the produced platelets are collected through the opening 11a of the holding member 11, the platelet producing apparatus 20 omits the step of separating megakaryocytes and platelets. Moreover, the platelet production apparatus 20 is set so that force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the suction pump 12. Thereby, the platelet production apparatus 20 can produce platelets efficiently.
  • the platelet production device 21 has a configuration in which the culture solution of the platelet production device 13 according to the third embodiment described above is circulated by the supply pump 4 and is stirred by the stirring device 19a, and is generated between charges. It is configured as a stirring charge system that produces platelets by Coulomb force.
  • the stirred charge type platelet production device 21 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 14, a collection tank 15, and a charge generation device 16.
  • the configuration of stirring the culture solution in the platelet production chamber 19 of the platelet production device 21 is the same as the configuration of the platelet production device 17 in the fourth embodiment described above.
  • the configuration for applying the Coulomb force using the electric charge charged to the megakaryocytes held by the holding member 11 of the platelet production device 21 is the same as the configuration of the platelet production device 13 in the third embodiment described above.
  • the agitation and suction type platelet production apparatus 21 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Furthermore, the platelet production device 21 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. Further, the platelet production device 21 is configured such that a Coulomb force is generated between the megakaryocyte charged with a negative charge by generating a positive charge with the charge generation device 16.
  • the platelet production device 21 produces platelets by applying Coulomb force in the direction of being drawn into the collection tank 15 to the megakaryocytes held by the holding member 14. Furthermore, since only the produced platelets are collected through the opening 11a of the holding member 14, the platelet producing apparatus 21 omits the step of separating megakaryocytes and platelets. In addition, the platelet production device 21 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the amount of charge generated by the charge generation device 16. Thereby, the platelet production apparatus 21 can produce platelets efficiently.
  • the platelet production device 1 and the platelet production device 10 are used to compensate for the shortage of the culture solution collected together with the platelets at the time of platelet production.
  • Between the platelet production chamber 19 and the collection tank 15 in the platelet production apparatus 21 and the physiological saline and the like are supplied to the storage tank 2 and the platelet production chambers 5 and 19 at a predetermined timing. It is good also as a structure.
  • a plurality of forces may be applied to the megakaryocyte by combining the modes for applying the force to the megakaryocyte in the first to sixth embodiments described above.
  • a force may be applied to the megakaryocyte by the flow of the liquid.
  • the platelet production apparatus 1, the platelet production apparatus 10, and the platelet production apparatus 13 are set so that a force appropriate for the production of platelets is applied to the megakaryocytes by further controlling the flow rate of the supply pump 4. Thereby, the platelet production apparatus 10 can produce platelets efficiently.
  • the collection pump 8 is provided on the downstream side of the holding member 6 and held.
  • the pressure inside the holding member 11 is lowered, and a pressure difference is generated between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 10. You may let them.
  • the megakaryocyte held by the holding member 6 is applied with a force that pushes in the flow direction from the collected liquid flowing in the collection flow path 7 to the portion of the megakaryocyte that is stuck in the opening 6a, and also produces platelets.
  • a force in the direction of being drawn into the collection channel 7 due to a pressure difference between the chamber 5 or the platelet production chamber 19 and the collection channel 7 is applied.
  • the megakaryocyte is applied with the force of the flow of the recovery liquid supplied by the recovery pump 8 and the suction force of the suction pump 12. Therefore, the platelet production device 21 can control the force applied to the megakaryocyte by controlling the flow rates of the supply pump 4 and the suction pump 12.
  • a force in a direction of being drawn into the holding member 6 may be generated by a Coulomb force generated between a negative charge charged in the megakaryocyte and a positive charge generated by the charge generation device 16. . Therefore, the platelet production device 21 can control the force applied to the megakaryocyte by controlling the flow rates of the supply pump 4 and the recovery pump 8 and the charge generation amount of the charge generation device 16.
  • the holding member 6, the holding member 11, and the holding member 14 in the first to sixth embodiments are formed of a nucleated cell-capturing material having a high ability to capture megakaryocytes that are nucleated cells to hold the megakaryocytes.
  • the structure to do may be sufficient.
  • capture materials polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane and other synthetic polymers, agarose, cellulose, cellulose acetate, chitin, chitosan, alginates and other natural polymers, Examples thereof include inorganic materials such as hydroxyapatite, glass, alumina and titania, and metals such as stainless steel, titanium and aluminum.
  • these capture materials can be used as they are, but they may be subjected to surface modification as necessary, for example, to increase platelet permeability or to selectively capture cells.
  • surface modification for example, to increase the platelet permeability, there can be mentioned a method of coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group proposed in WO 87/05812.
  • the platelet production apparatus cultivates megakaryocyte progenitor cells derived from iPS cells under predetermined conditions to produce mature megakaryocytes.
  • You may comprise as the platelet production system 22 provided with the concentration apparatus 26 which concentrates. By comprising in this way, a platelet formulation can be continuously manufactured from the megakaryocyte precursor cell induced

Abstract

Provided are a platelet production device and a platelet production method, whereby platelets can be efficiently produced. Specifically speaking, the platelet production device comprises: a platelet production chamber (5) that is a container storing a megakaryocyte-containing liquid; a feed pump (4) that is a feeding means feeding a liquid culture medium, said liquid culture medium being the megakaryocyte-containing liquid, to the platelet production chamber (5); a retention member (6) that is a retaining means provided in a part of the platelet production chamber (5) for retaining megakaryocytes; and a recovery pump (8) that is an external force-applying means applying an external force, said external force being generated by the flow of the recovered liquid, to the megakaryocytes retained by the retention member (6) and thus allowing the megakaryocytes to produce platelets.

Description

血小板産生装置および血小板産生方法Platelet production apparatus and platelet production method
　本発明は、血小板産生装置および血小板産生方法に関する。詳しくは、ｉＰＳ細胞から誘導された巨核球から血小板を産生させる装置および方法に関する。 The present invention relates to a platelet production apparatus and a platelet production method. More specifically, the present invention relates to an apparatus and method for producing platelets from megakaryocytes derived from iPS cells.
　従来、血小板は、比重の差を利用して分離する遠心分離法によって血液中から分離されている。例えば特許文献１の如くである。特許文献１に記載の遠心分離機は、血液が流入された遠心分離機の遠心ボウルが高速回転することで、遠心ボウルの外縁部に形成されている貯血空間内において各血液細胞の比重毎に、血漿層、バフィーコート層および赤血球層に分離される。そして、バフィーコート層から血小板を遠心分離によって分離させる。 Conventionally, platelets are separated from blood by a centrifugal separation method using a difference in specific gravity. For example, it is like patent document 1. The centrifugal separator described in Patent Document 1 is provided for each specific gravity of each blood cell in the blood storage space formed at the outer edge of the centrifugal bowl by rotating the centrifugal bowl of the centrifuge into which blood has flowed at high speed. , Separated into plasma layer, buffy coat layer and red blood cell layer. Then, the platelets are separated from the buffy coat layer by centrifugation.
　一方、ｉＰＳ細胞から誘導された巨核球から血小板を産生させる場合、血小板の産生後に血小板を産生せずに残留した有核細胞である巨核球を除去する必要がある。しかし、巨核球と血小板とは比重が近いため、遠心分離法によって巨核球と血小板とを分離するためには、大きな遠心加速度を付加する必要がある。このため、遠心分離法によって巨核球と血小板とが分離できても、大きな遠心加速度により破壊される血小板の割合が増大して血小板の回収率が低下する問題があった。 On the other hand, when platelets are produced from megakaryocytes derived from iPS cells, it is necessary to remove megakaryocytes, which are nucleated cells remaining after platelet production without producing platelets. However, since the specific gravity of megakaryocytes and platelets is close, in order to separate megakaryocytes and platelets by centrifugation, it is necessary to add a large centrifugal acceleration. For this reason, even if megakaryocytes and platelets can be separated by the centrifugal separation method, there is a problem that the ratio of platelets destroyed by a large centrifugal acceleration increases and the platelet recovery rate decreases.
特開平９－１０８５９４号公報JP-A-9-108594
　本発明の目的は、効率よく血小板を産生することができる血小板産生装置および血小板産生方法を提供することである。 An object of the present invention is to provide a platelet production apparatus and a platelet production method capable of efficiently producing platelets.
　本発明の解決しようとする課題は以上の如くであり、次にこの課題を解決するための手段を説明する。 The problems to be solved by the present invention are as described above. Next, means for solving the problems will be described.
　即ち、本発明は、巨核球が含まれる液体を貯留する容器と、容器に巨核球が含まれる液体を供給する供給手段と、容器の一部にもうけられ、巨核球を保持する保持手段と、前記保持手段により保持された巨核球に外力を加えて血小板を産生させる外力付与手段と、を具備するものである。 That is, the present invention is a container for storing a liquid containing megakaryocytes, a supply means for supplying a liquid containing megakaryocytes in the container, a holding means provided in a part of the container and holding the megakaryocytes, An external force applying means for applying an external force to the megakaryocyte held by the holding means to produce platelets.
　本発明は、前記保持手段に前記血小板が通過できて前記巨核球が通過できない大きさの開口部が形成され、開口部の一側に巨核球の一部分がはまり込むことで巨核球を保持するものである。 In the present invention, the holding means is formed with an opening having a size that allows the platelets to pass therethrough and the megakaryocytes cannot pass through, and a megakaryocyte is held in one part of the opening by holding the megakaryocyte. It is.
　本発明は、前記外力付与手段が前記開口部の他側から流体の流れにより前記巨核球に外力を加えるものである。 In the present invention, the external force applying means applies an external force to the megakaryocyte by a fluid flow from the other side of the opening.
　本発明は、前記外力付与手段が前記開口部の一側と他側との間に圧力差を生じさせることにより前記巨核球に外力を加えるものである。 In the present invention, the external force applying means applies an external force to the megakaryocyte by creating a pressure difference between one side and the other side of the opening.
　本発明は、前記外力付与手段が巨核球に帯電している電荷と異なる電荷を前記開口部の他側に生じさせることにより前記巨核球に外力を加えるものである。 In the present invention, the external force applying means applies an external force to the megakaryocyte by generating a charge on the other side of the opening that is different from the charge charged on the megakaryocyte.
　本発明は、巨核球を保持し、外部から巨核球に力を加えて、巨核球から血小板を産生させるものである。 The present invention holds megakaryocytes and applies force to the megakaryocytes from the outside to produce platelets from the megakaryocytes.
　本発明の効果として、以下に示すような効果を奏する。 As the effects of the present invention, the following effects are obtained.
　本発明においては、巨核球を保持した状態で血小板が産生されるので巨核球と血小板とを分離する工程が省かれる。これにより、効率よく血小板を産生することができる。 In the present invention, since platelets are produced while holding megakaryocytes, the step of separating megakaryocytes and platelets is omitted. Thereby, platelets can be produced efficiently.
　本発明においては、巨核球に加わる力が任意の値に設定されるとともに、保持手段の開口部の他側から血小板が産生される。これにより、効率よく血小板を産生することができる。 In the present invention, the force applied to the megakaryocyte is set to an arbitrary value, and platelets are produced from the other side of the opening of the holding means. Thereby, platelets can be produced efficiently.
本発明の第一実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 1st embodiment of this invention. （ａ）本発明の第一実施形態に係る血小板産生装置における血小板産生室の構成を示す概念図（ｂ）同じく孔が形成されている保持部材を示す部分拡大図（ｃ）同じくスリットが形成されている保持部材を示す部分拡大図。(A) The conceptual diagram which shows the structure of the platelet production chamber in the platelet production apparatus which concerns on 1st embodiment of this invention (b) The partial enlarged view which similarly shows the holding member in which the hole is formed (c) The slit is similarly formed. The elements on larger scale which show the holding member. （ａ）本発明の第一実施形態に係る血小板産生装置において巨核球が保持部材に接触した状態を示す模式図（ｂ）同じく巨核球が保持部材の開口部に入り込んだ状態を示す模式図（ｃ）同じく巨核球が血小板を産生している状態を示す模式図。(A) Schematic diagram showing the state in which the megakaryocyte is in contact with the holding member in the platelet production apparatus according to the first embodiment of the present invention (b) Schematic diagram showing the state in which the megakaryocyte has also entered the opening of the holding member ( c) A schematic diagram showing a state in which megakaryocytes are also producing platelets. 本発明の第二実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 2nd embodiment of this invention. （ａ）本発明の第二実施形態に係る血小板産生装置における血小板産生室の構成を示す概念図（ｂ）同じく巨核球が血小板を産生している状態を示す模式図。(A) The conceptual diagram which shows the structure of the platelet production chamber in the platelet production apparatus which concerns on 2nd embodiment of this invention (b) The schematic diagram which shows the state from which the megakaryocyte is also producing platelets. 本発明の第三実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 3rd embodiment of this invention. （ａ）本発明の第三実施形態に係る血小板産生装置における血小板産生室の構成を示す概念図（ｂ）同じく巨核球が血小板を産生している状態を示す模式図。(A) The conceptual diagram which shows the structure of the platelet production chamber in the platelet production apparatus which concerns on 3rd embodiment of this invention (b) The schematic diagram which shows the state in which megakaryocytes are producing platelets similarly. 本発明の第四実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 4th embodiment of this invention. 本発明の第五実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 5th embodiment of this invention. 本発明の第六実施形態に係る血小板産生装置の全体構成を示す概略図。Schematic which shows the whole structure of the platelet production apparatus which concerns on 6th embodiment of this invention. 本発明に係る血小板産生装置を含む血小板産生システムの全体構成を示す概略図。Schematic which shows the whole structure of the platelet production system containing the platelet production apparatus which concerns on this invention.
　まず、図１と図２とを用いて、本発明に係る血小板産生装置の第一実施形態である血小板産生装置１について説明する。なお、以下の各実施形態において、血小板産生装置を構成する各部材は、特に記載がない限り、非反応性ポリマー、生物親和性金属、ガラス等のうち用途や強度から適切な材料によって構成されている。具体的には、非反応性ポリマーとしては、アクリロニトリルブタジエンスチレンターポリマー等のアクリロ二トリルポリマー、ポリ塩化ビニル等のハロゲン化ポリマー、ポリアミド、ポリイミドポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン等である。また、生物親和性金属としては、ステンレス鋼、チタン、白金およびこれらの合金、コバルトクロミウム合金等である。 First, the platelet production apparatus 1 which is the first embodiment of the platelet production apparatus according to the present invention will be described with reference to FIGS. 1 and 2. In each of the following embodiments, each member constituting the platelet production device is made of an appropriate material from the application and strength among non-reactive polymer, biocompatible metal, glass and the like unless otherwise specified. Yes. Specifically, examples of the non-reactive polymer include acrylonitrile polymers such as acrylonitrile butadiene styrene terpolymer, halogenated polymers such as polyvinyl chloride, polyamide, polyimide polycarbonate, polyethylene, polypropylene, and polystyrene. Examples of the biocompatible metal include stainless steel, titanium, platinum and alloys thereof, and cobalt chromium alloy.
　図１に示すように、血小板産生装置１は、巨核球から血小板を産生し回収するものである。血小板産生装置１は、巨核球が含まれる液体と血小板が含まれる液体とを共に循環させながら血小板を産生する両循環方式として構成されている。血小板産生装置１は、貯留タンク２、循環流路３、供給ポンプ４、血小板産生室５、保持部材６、回収流路７、回収ポンプ８および回収タンク９を具備している。 As shown in FIG. 1, the platelet production apparatus 1 produces and collects platelets from megakaryocytes. The platelet production apparatus 1 is configured as a double circulation system that produces platelets while circulating a liquid containing megakaryocytes and a liquid containing platelets together. The platelet production apparatus 1 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 6, a collection channel 7, a collection pump 8, and a collection tank 9.
　貯留タンク２は、成熟した巨核球と成熟した巨核球の一部から産生された血小板とが含まれる液体（以下、単に「培養液」と記す）を保持するものである。貯留タンク２は、外部から培養液を供給可能に構成されている。具体的には、貯留タンク２は、後述の培養装置２３等に接続され、培養装置２３等で製造された培養液が供給されるように構成されている。 The storage tank 2 holds a liquid (hereinafter simply referred to as “culture medium”) containing mature megakaryocytes and platelets produced from a part of the mature megakaryocytes. The storage tank 2 is configured to be able to supply a culture solution from the outside. Specifically, the storage tank 2 is connected to a culture device 23 and the like which will be described later, and is configured to be supplied with a culture solution produced by the culture device 23 and the like.
　循環流路３は、培養液を循環させるための流路である。循環流路３は、非反応性ポリマー、生物親和性金属、ガラス等からなる管から構成されている。循環流路３は、供給ポンプ４を介して貯留タンク２と血小板産生室５とを接続するように構成されている。また、循環流路３は、血小板産生室５と貯留タンク２とを接続するように構成されている。すなわち、貯留タンク２内の培養液は、供給ポンプ４によって循環流路３を通じて血小板産生室５に供給されるように構成されている。そして、血小板産生室５に供給された培養液は、循環流路３を通じて再び貯留タンク２に戻されるように構成されている。 The circulation channel 3 is a channel for circulating the culture solution. The circulation channel 3 is composed of a tube made of a non-reactive polymer, a biocompatible metal, glass or the like. The circulation channel 3 is configured to connect the storage tank 2 and the platelet production chamber 5 via the supply pump 4. The circulation channel 3 is configured to connect the platelet production chamber 5 and the storage tank 2. That is, the culture solution in the storage tank 2 is configured to be supplied to the platelet production chamber 5 through the circulation channel 3 by the supply pump 4. Then, the culture solution supplied to the platelet production chamber 5 is configured to be returned to the storage tank 2 again through the circulation channel 3.
　供給手段である供給ポンプ４は、貯留タンク２内の培養液を循環流路３に供給するものである。供給ポンプ４は、例えばローラーポンプまたは連続シリンジポンプから構成されるがこれに限定するものではない。供給ポンプ４は、循環流路３の途中部に設けられている。これにより、供給ポンプ４は、貯留タンク２内の培養液を循環流路３に供給することで培養液を貯留タンク２と血小板産生室５との間で循環させるように構成されている。供給ポンプ４は、任意の流量で培養液を循環流路３に供給できるように構成されている。なお、本実施形態において、血小板産生装置１は、供給ポンプ４によって培養液を供給する構成としたがこれに限定されるものではない。 The supply pump 4 serving as a supply means supplies the culture solution in the storage tank 2 to the circulation channel 3. Although the supply pump 4 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this. The supply pump 4 is provided in the middle of the circulation channel 3. Thus, the supply pump 4 is configured to circulate the culture solution between the storage tank 2 and the platelet production chamber 5 by supplying the culture solution in the storage tank 2 to the circulation channel 3. The supply pump 4 is configured so that the culture solution can be supplied to the circulation channel 3 at an arbitrary flow rate. In the present embodiment, the platelet production device 1 is configured to supply the culture solution by the supply pump 4, but is not limited thereto.
　容器である血小板産生室５は、巨核球から血小板を産生させる空間を構成するものである。血小板産生室５は、非反応性ポリマーまたは生物親和性金属等から形成される容器である。血小板産生室５は、循環流路３の途中部に設けられている。つまり、血小板産生室５は、循環流路３を通じて貯留タンク２から培養液が供給されるとともに、血小板産生室５内の培養液が貯留タンク２に戻されるように構成されている。 The platelet production chamber 5, which is a container, constitutes a space for producing platelets from megakaryocytes. The platelet production chamber 5 is a container formed from a non-reactive polymer or a biocompatible metal. The platelet production chamber 5 is provided in the middle of the circulation channel 3. That is, the platelet production chamber 5 is configured so that the culture solution is supplied from the storage tank 2 through the circulation channel 3 and the culture solution in the platelet production chamber 5 is returned to the storage tank 2.
　保持手段である保持部材６は、巨核球を保持するものである。保持部材６は、非反応性ポリマーまたは生物親和性金属等からなる薄板（もしくはフィルター）の一側面から他側面に連通するように複数の開口部６ａが形成されている（図２参照）。保持部材６は、血小板産生室５にその空間の一部を仕切るように設けられている。つまり、保持部材６の一側面は、血小板産生室５の内部に配置され、培養液に含まれている巨核球が開口部６ａに接触するように構成されている。また、保持部材６の他側面は、血小板産生室５の内部における回収流路７の一部を構成している。 The holding member 6 which is a holding means holds a megakaryocyte. The holding member 6 is formed with a plurality of openings 6a so as to communicate from one side surface to the other side surface of a thin plate (or filter) made of a non-reactive polymer or a biocompatible metal (see FIG. 2). The holding member 6 is provided in the platelet production chamber 5 so as to partition a part of the space. That is, one side surface of the holding member 6 is arranged inside the platelet production chamber 5 so that megakaryocytes contained in the culture solution are in contact with the opening 6a. In addition, the other side surface of the holding member 6 constitutes a part of the collection flow path 7 in the platelet production chamber 5.
　具体的には、図２（ａ）に示すように、保持部材６は、薄板等を中空の管状に形成して構成されている。保持部材６は、一側面である管の外側と他側面である管の内側とを連通するように開口部６ａが形成されている。開口部６ａは、例えば、図２（ｂ）に示す孔である場合における直径Ｄ１、または図２（ｃ）に示すスリットである場合におけるスリットの幅Ｄ２が血小板の通過できる大きさ（３μｍから４μｍ以上）、かつ巨核球の一部分がはまり込んで開口部６ａの一側に保持される大きさに構成されている。なお、ｉＰＳ細胞から誘導される巨核球の大きさは、その培養過程により変化する。したがって、そのときの巨核球の大きさに基づいて、孔の直径Ｄ１やスリットの幅Ｄ２を決定することが望ましい。保持部材６は、血小板産生室５に配置され、その両方の端部に回収流路７が接続されている。 Specifically, as shown in FIG. 2A, the holding member 6 is configured by forming a thin plate or the like into a hollow tubular shape. The holding member 6 has an opening 6a formed so as to communicate the outer side of the tube that is one side surface and the inner side of the tube that is the other side surface. For example, the opening 6a has a diameter D3 in the case of the hole shown in FIG. 2B or a slit width D2 in the case of the slit shown in FIG. And a size that allows a part of the megakaryocyte to fit and be held on one side of the opening 6a. The size of megakaryocytes derived from iPS cells varies depending on the culture process. Therefore, it is desirable to determine the hole diameter D1 and the slit width D2 based on the size of the megakaryocyte at that time. The holding member 6 is disposed in the platelet production chamber 5, and a recovery channel 7 is connected to both ends thereof.
　なお、本実施形態において、保持部材６の断面形状は、円形に限定されるものではなく、例えば、矩形や楕円の断面形状に形成してもよい。また、血小板産生室５に設けられる保持部材６の大きさや数は、血小板産生室５内における培養液の流れを阻害しない程度であれば特に限定されるものではない。例えば、開口部６ａで保持する巨核球の数を増やすために、保持部材６の管の大きさをできるだけ大きくしたり複数の保持部材６を血小板産生室５に設けたりして保持部材６の表面積を増大させてもよい。 In the present embodiment, the cross-sectional shape of the holding member 6 is not limited to a circular shape, and may be a rectangular or elliptical cross-sectional shape, for example. Further, the size and number of the holding members 6 provided in the platelet production chamber 5 are not particularly limited as long as they do not hinder the flow of the culture solution in the platelet production chamber 5. For example, in order to increase the number of megakaryocytes held in the opening 6a, the surface area of the holding member 6 is increased by increasing the size of the tube of the holding member 6 as much as possible or providing a plurality of holding members 6 in the platelet production chamber 5. May be increased.
　図１と図２とに示すように、回収流路７は、培養液に含まれる血小板と巨核球からすでに産生された血小板とを回収するための液体（以下、単に「回収液」と記す）が流れる流路である。回収流路７は、保持部材６の一方（入口側）の端部と回収タンク９とを接続するように構成されている。また、回収流路７は、保持部材６の他方（出口側）の端部と回収タンク９とを接続するように構成されている。 As shown in FIGS. 1 and 2, the recovery channel 7 is a liquid for recovering platelets contained in the culture medium and platelets already produced from megakaryocytes (hereinafter simply referred to as “recovery liquid”). It is a flow path through which. The collection channel 7 is configured to connect one end (inlet side) end of the holding member 6 and the collection tank 9. The recovery channel 7 is configured to connect the other end (exit side) end of the holding member 6 and the recovery tank 9.
　外力付与手段である回収ポンプ８は、回収タンク９内の回収液を回収流路７に供給するものである。供給ポンプ４は、例えばローラーポンプまたは連続シリンジポンプから構成されるがこれに限定するものではない。回収ポンプ８は、保持部材６の一方（入口側）の端部と回収タンク９とを接続する回収流路７の途中部に設けられている。これにより、回収ポンプ８は、回収タンク９内の回収液を回収流路７に供給することで保持部材６と回収タンク９との間で回収液を循環させるように構成されている。回収ポンプ８は、任意の流量で回収液を回収流路７に供給できるように構成されている。なお、本実施形態において、回収ポンプ８によって回収液を供給する構成としたがこれに限定されるものではない。 The recovery pump 8 which is an external force applying means supplies the recovery liquid in the recovery tank 9 to the recovery flow path 7. Although the supply pump 4 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this. The recovery pump 8 is provided in the middle of the recovery flow path 7 that connects one end (inlet side) of the holding member 6 and the recovery tank 9. Thus, the recovery pump 8 is configured to circulate the recovery liquid between the holding member 6 and the recovery tank 9 by supplying the recovery liquid in the recovery tank 9 to the recovery flow path 7. The recovery pump 8 is configured to supply the recovery liquid to the recovery flow path 7 at an arbitrary flow rate. In the present embodiment, the recovery liquid is supplied by the recovery pump 8, but the present invention is not limited to this.
　このように構成される両循環方式の血小板産生装置１は、貯留タンク２内の培養液が供給ポンプ４によって循環流路３を通じて血小板産生室５に供給されるように構成されている（図２（ａ）太矢印参照）。さらに、血小板産生装置１は、血小板産生室５に供給された培養液が循環流路３を通じて貯留タンク２に排出されるように構成されている。すなわち、血小板産生装置１は、貯留タンク２と血小板産生室５との間で培養液が循環するように構成されている。また、血小板産生装置１は、回収タンク９内の回収液が回収ポンプ８によって回収流路７を通じて保持部材６の内部に供給されるように構成されている。さらに、血小板産生装置１は、保持部材６の内部に供給された回収液が回収流路７を通じて回収タンク９に排出されるように構成されている。すなわち、血小板産生装置１は、回収タンク９と保持部材６との間で回収液が循環するように構成されている（図２（ａ）白塗り矢印参照）。 The double-circulation type platelet production apparatus 1 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 5 through the circulation channel 3 by the supply pump 4 (FIG. 2). (See a thick arrow). Further, the platelet production device 1 is configured such that the culture solution supplied to the platelet production chamber 5 is discharged to the storage tank 2 through the circulation channel 3. That is, the platelet production apparatus 1 is configured so that the culture solution circulates between the storage tank 2 and the platelet production chamber 5. The platelet production apparatus 1 is configured such that the collected liquid in the collection tank 9 is supplied to the inside of the holding member 6 through the collection channel 7 by the collection pump 8. Further, the platelet production device 1 is configured such that the collected liquid supplied into the holding member 6 is discharged to the collection tank 9 through the collection channel 7. That is, the platelet production apparatus 1 is configured such that the collected liquid circulates between the collection tank 9 and the holding member 6 (see the white arrow in FIG. 2A).
　次に、図１と図３とを用いて、血小板産生装置１における血小板産生方法の態様について具体的に説明する。なお、以下の各実施形態において、保持部材６の開口部６ａは孔状に形成されているものとして説明する。 Next, the embodiment of the platelet production method in the platelet production apparatus 1 will be specifically described with reference to FIGS. In the following embodiments, the description will be made assuming that the opening 6a of the holding member 6 is formed in a hole shape.
　図１に示すように、血小板産生装置１は、供給ポンプ４によって、貯留タンク２内の培養液を循環流路３に供給する。これにより、培養液に含まれる巨核球と巨核球からすでに産生された血小板とが循環流路３を通じて血小板産生室５に供給される。 As shown in FIG. 1, the platelet production apparatus 1 supplies the culture solution in the storage tank 2 to the circulation channel 3 by the supply pump 4. Thereby, the megakaryocytes contained in the culture solution and the platelets already produced from the megakaryocytes are supplied to the platelet production chamber 5 through the circulation channel 3.
　図３（ａ）に示すように、培養液とともに血小板産生室５に供給された巨核球は、一部の巨核球Ａが保持部材６に接触する。図３（ｂ）に示すように、保持部材６に接触した巨核球Ａは、保持部材６に設けられている開口部６ａの一側にその一部分がはまり込んで保持部材６に保持される。巨核球の粒径は、開口部６ａの直径Ｄ１（図２（ｂ）参照）よりも大きいので開口部６ａを通過することができない。一方、培養液とともに血小板産生室５に供給された血小板は、一部の血小板が保持部材６の開口部６ａに入り込む。血小板の粒径は、開口部６ａの直径Ｄ１よりも小さいので開口部６ａを通過することができる。すなわち、保持部材６に接触した一部の巨核球Ａと血小板とのうち、血小板のみが開口部６ａから保持部材６の内部に入り込む。 As shown in FIG. 3A, some megakaryocytes A of the megakaryocytes supplied to the platelet production chamber 5 together with the culture solution come into contact with the holding member 6. As shown in FIG. 3B, the megakaryocyte A in contact with the holding member 6 is held by the holding member 6 with a part of the megakaryocyte A fitted into one side of the opening 6 a provided in the holding member 6. Since the particle size of the megakaryocyte is larger than the diameter D1 (see FIG. 2B) of the opening 6a, it cannot pass through the opening 6a. On the other hand, some of the platelets supplied to the platelet production chamber 5 together with the culture solution enter the opening 6 a of the holding member 6. Since the particle size of platelets is smaller than the diameter D1 of the opening 6a, it can pass through the opening 6a. That is, only some platelets enter the inside of the holding member 6 from the opening 6a out of some megakaryocytes A and platelets in contact with the holding member 6.
　培養液に含まれる巨核球のうち、保持部材６に接触しなかった巨核球や保持部材６に接触したが開口部６ａにはまり込まなかった巨核球Ｂは、培養液とともに血小板産生室５から排出されて貯留タンク２内に戻される。同様にして、培養液に含まれる血小板のうち、開口部６ａを通過して保持部材６の内部に入り込まなかった血小板は、培養液とともに血小板産生室５から排出されて貯留タンク２内に戻される。貯留タンク２に戻された巨核球と血小板とは、供給ポンプ４で他の巨核球と血小板とともに再び循環流路３を通じて血小板産生室５に供給される。 Among megakaryocytes contained in the culture solution, megakaryocytes that did not contact the holding member 6 and megakaryocytes B that contacted the holding member 6 but did not get stuck in the opening 6a were discharged from the platelet production chamber 5 together with the culture solution. And returned to the storage tank 2. Similarly, among the platelets contained in the culture solution, the platelets that have passed through the opening 6a and have not entered the holding member 6 are discharged together with the culture solution from the platelet production chamber 5 and returned to the storage tank 2. . The megakaryocytes and platelets returned to the storage tank 2 are supplied again to the platelet production chamber 5 through the circulation channel 3 together with other megakaryocytes and platelets by the supply pump 4.
　図３（ｂ）に示すように、保持部材６に保持されている巨核球Ａには、開口部６ａにはまり込んでいる部分に外力付与手段である回収ポンプ８によって回収流路７内を循環している回収液からその流れ方向に押す力が加わる（図３細矢印参照）。この際、血小板産生室５内の培養液は常に流れが発生しているので、培養液中の他の巨核球が堆積することによって巨核球Ａが保持部材６に押し付けられることがない。つまり、保持されている巨核球Ａには、回収ポンプ８が供給する回収液の流れによる力のみが加わっている。従って、血小板産生装置１は、回収ポンプ８の流量を制御することで巨核球Ａに加わる力を制御することができる。回収液の流れ方向に押された巨核球Ａには、回収液の流れによる力が加わっている箇所や開口部６ａとの接触部分にせん断応力が生じる。 As shown in FIG. 3 (b), the megakaryocyte A held by the holding member 6 is circulated in the recovery flow path 7 by a recovery pump 8 which is an external force applying means in a portion that is fitted in the opening 6a. The pushing force is applied from the collected liquid in the flow direction (see thin arrows in FIG. 3). At this time, since the culture solution in the platelet production chamber 5 is always flowing, the megakaryocytes A are not pressed against the holding member 6 due to the accumulation of other megakaryocytes in the culture solution. That is, only the force due to the flow of the recovery liquid supplied by the recovery pump 8 is applied to the held megakaryocyte A. Therefore, the platelet production apparatus 1 can control the force applied to the megakaryocyte A by controlling the flow rate of the collection pump 8. In the megakaryocyte A pushed in the flow direction of the collected liquid, a shear stress is generated at a position where a force due to the flow of the collected liquid is applied and a contact portion with the opening 6a.
　図３（ｃ）に示すように、巨核球Ａは、回収液の流れによってせん断応力が発生している箇所を起点として巨核球Ａを形成している細胞質の一部分がせん断される。つまり、巨核球Ａは、回収液の流れによる力によってその細胞質の一部分が分離して血小板Ｃが産生される。産生された血小板は、回収液中に混入して回収液とともに回収流路７を通じて回収タンク９に搬送される。 As shown in FIG. 3 (c), in the megakaryocyte A, a part of the cytoplasm forming the megakaryocyte A is sheared starting from a location where shear stress is generated by the flow of the recovered liquid. That is, in the megakaryocyte A, a part of its cytoplasm is separated by the force caused by the flow of the recovered liquid, and platelets C are produced. The produced platelets are mixed in the recovery liquid and conveyed to the recovery tank 9 through the recovery flow path 7 together with the recovery liquid.
　保持部材６に保持されている巨核球は、回収液の流れによる力が加わっている間、血小板の産生が続けられる。保持部材６から離間した巨核球は、血小板の産生を停止し、培養液とともに血小板産生室５から排出されて貯留タンク２内に戻される。また、血小板産生室５内において他の保持部材６に再び保持された巨核球は、血小板の産生を再開する。血小板産生室５から排出されて貯留タンク２に戻された巨核球と血小板とは、供給ポンプ４で他の巨核球と血小板とともに再び循環流路３を通じて血小板産生室５に供給される。 The megakaryocytes held by the holding member 6 continue to produce platelets while the force due to the flow of the collected liquid is applied. Megakaryocytes separated from the holding member 6 stop producing platelets, are discharged from the platelet production chamber 5 together with the culture solution, and are returned to the storage tank 2. Further, megakaryocytes held again by the other holding member 6 in the platelet production chamber 5 resume production of platelets. Megakaryocytes and platelets discharged from the platelet production chamber 5 and returned to the storage tank 2 are supplied again to the platelet production chamber 5 through the circulation channel 3 together with other megakaryocytes and platelets by the supply pump 4.
　このように構成することで、血小板産生装置１は、保持部材６に保持されている巨核球に回収液の流れによる力を加えることで血小板を産生させる。さらに、血小板産生装置１は、産生された血小板のみが回収液に混入して回収されるので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置１は、回収ポンプ８の流量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置１は、効率よく血小板を産生することができる。 With this configuration, the platelet production device 1 produces platelets by applying force due to the flow of the collected liquid to the megakaryocytes held by the holding member 6. Furthermore, since the platelet production apparatus 1 collects only the produced platelets and collects them in the collection solution, the step of separating megakaryocytes and platelets is omitted. Moreover, the platelet production apparatus 1 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the collection pump 8. Thereby, the platelet production apparatus 1 can produce platelets efficiently.
　以下では、図４と図５とを用いて、本発明に係る血小板産生装置の第二実施形態における血小板産生装置１０について説明する。なお、以下の実施形態において、既に説明した実施形態と同様の点に関してはその具体的説明を省略し、相違する部分を中心に説明する。 Hereinafter, the platelet production apparatus 10 in the second embodiment of the platelet production apparatus according to the present invention will be described with reference to FIGS. 4 and 5. In the following embodiments, the same points as those of the above-described embodiments will not be specifically described, and different portions will be mainly described.
　図４に示すように、血小板産生装置１０は、巨核球が含まれる液体を循環させながら吸引（圧力差）により血小板を産生する循環吸引方式として構成されている。血小板産生装置１０は、貯留タンク２、循環流路３、供給ポンプ４、血小板産生室５、保持部材１１、回収流路７、吸引ポンプ１２および回収タンク９を具備している。 As shown in FIG. 4, the platelet production apparatus 10 is configured as a circulating suction system that produces platelets by suction (pressure difference) while circulating a liquid containing megakaryocytes. The platelet production apparatus 10 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 11, a collection channel 7, a suction pump 12, and a collection tank 9.
　血小板産生装置１０の培養液を循環させる構成は、前述の第一実施形態にかかる血小板産生装置１の構成と同一である。 The configuration for circulating the culture solution of the platelet production device 10 is the same as the configuration of the platelet production device 1 according to the first embodiment described above.
　図４と図５（ａ）とに示すように、保持手段である保持部材１１は、一方の端部を閉塞させた中空の管状に形成されている。保持部材１１は、一側面である管の外側と他側面である管の内側とを連通するように開口部１１ａが形成されている。保持部材１１は、血小板産生室５に配置される。また、保持部材１１は、その他方の端部が回収流路７を介して回収タンク９に接続されている。なお、本実施形態において、貯留タンク２内に設けられる保持部材１１の大きさや数は、特に限定されるものではない。例えば、開口部１１ａで保持する巨核球の数を増やすために、保持部材１１の管の大きさをできるだけ大きくしたり複数の保持部材１１を血小板産生室５に設けたりして保持部材１１の表面積を増大させてもよい。 As shown in FIG. 4 and FIG. 5 (a), the holding member 11 as the holding means is formed in a hollow tubular shape with one end closed. The holding member 11 has an opening portion 11a formed so as to communicate the outside of the tube that is one side surface and the inside of the tube that is the other side surface. The holding member 11 is disposed in the platelet production chamber 5. The other end of the holding member 11 is connected to the recovery tank 9 via the recovery flow path 7. In the present embodiment, the size and number of the holding members 11 provided in the storage tank 2 are not particularly limited. For example, in order to increase the number of megakaryocytes held in the opening 11a, the surface area of the holding member 11 is increased by increasing the size of the tube of the holding member 11 as much as possible or by providing a plurality of holding members 11 in the platelet production chamber 5. May be increased.
　外力付与手段である吸引ポンプ１２は、回収流路７内の圧力を循環流路３の圧力よりも低くするものである。吸引ポンプ１２は、例えばローラーポンプまたは連続シリンジポンプから構成されるがこれに限定するものではない。吸引ポンプ１２は、保持部材１１と回収タンク９とを接続する回収流路７の途中部に設けられている。これにより、吸引ポンプ１２は、回収流路７の内部を吸引することで、回収流路７に接続されている保持部材１１の内部の圧力を低下させることができる（図５（ａ）白塗り矢印参照）。すなわち、血小板産生装置１０は、保持部材１１の内部の圧力と保持部材１１の外部である血小板産生室５の圧力とに圧力差を生じさせるように構成されている。吸引ポンプ１２は、その流量を変更することによって回収流路７の圧力を任意の圧力に設定することができる。 The suction pump 12, which is an external force applying means, makes the pressure in the recovery channel 7 lower than the pressure in the circulation channel 3. Although the suction pump 12 is comprised, for example from a roller pump or a continuous syringe pump, it is not limited to this. The suction pump 12 is provided in the middle of the recovery flow path 7 that connects the holding member 11 and the recovery tank 9. Thereby, the suction pump 12 can reduce the pressure inside the holding member 11 connected to the recovery flow path 7 by sucking the inside of the recovery flow path 7 (FIG. 5A) See arrow). That is, the platelet production apparatus 10 is configured to generate a pressure difference between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 5 that is outside the holding member 11. The suction pump 12 can set the pressure of the recovery flow path 7 to an arbitrary pressure by changing the flow rate.
　このように構成される循環吸引方式の血小板産生装置１０は、貯留タンク２と血小板産生室５との間で培養液が循環するように構成されている。また、血小板産生装置１０は、吸引ポンプ１２によって保持部材１１の内部の圧力と保持部材１１の外部である血小板産生室５の圧力との間に圧力差が生じるように構成されている。 The circulatory suction type platelet production apparatus 10 configured as described above is configured so that the culture medium circulates between the storage tank 2 and the platelet production chamber 5. The platelet production apparatus 10 is configured such that a pressure difference is generated between the pressure inside the holding member 11 and the pressure in the platelet production chamber 5 outside the holding member 11 by the suction pump 12.
　次に、図４と図５とを用いて、血小板産生装置１０における血小板産生方法の態様について具体的に説明する。 Next, the embodiment of the platelet production method in the platelet production apparatus 10 will be specifically described with reference to FIGS. 4 and 5.
　図５（ａ）に示すように、培養液とともに血小板産生室５に供給された巨核球と巨核球からすでに産生された血小板とは（太矢印参照）、一部の巨核球と血小板とが保持部材１１に設けられている開口部１１ａの一側にその一部分がはまり込んで保持部材１１に保持される。また、図５（ｂ）に示すように、保持部材１１は、血小板産生室５の内部の圧力と回収流路７の内部の圧力との圧力差により、開口部１１ａに血小板産生室５内の培養液を回収流路７内に吸引する力が生じる（細矢印参照）。開口部１１ａに生じている吸引力が及ぶ範囲にある巨核球および血小板は、開口部１１ａに向かって吸い寄せられる。開口部１１ａに吸い寄せられた一部の巨核球Ａは、開口部１１ａの一側にその一部分がはまり込んで保持部材１１に保持される。開口部１１ａに吸い寄せられた一部の血小板は、開口部１１ａから保持部材１１の内部に入り込む。 As shown in FIG. 5 (a), megakaryocytes supplied to the platelet production chamber 5 together with the culture solution and platelets already produced from the megakaryocytes (see thick arrows) are retained by some megakaryocytes and platelets. Part of the opening 11 a provided in the member 11 is fitted into one side and held by the holding member 11. Further, as shown in FIG. 5B, the holding member 11 has an opening 11a in the platelet production chamber 5 due to a pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the recovery flow path 7. A force for sucking the culture solution into the collection channel 7 is generated (see thin arrows). Megakaryocytes and platelets within the range of the suction force generated in the opening 11a are sucked toward the opening 11a. A part of the megakaryocytes A sucked into the opening portion 11a is fitted into one side of the opening portion 11a and held by the holding member 11. Some platelets sucked into the opening 11a enter the inside of the holding member 11 from the opening 11a.
　保持部材１１に保持されている巨核球Ａには、外力付与手段である吸引ポンプ１２によって生じた血小板産生室５と回収流路７との圧力差により回収流路７内に引き込まれる方向の力が加わる。すなわち、巨核球は、保持部材１１の内部に引き込まれる力によって保持部材１１に押し付けられている。この際、巨核球Ａには、血小板産生室５の内部の圧力と回収流路７の内部の圧力との圧力差による力（吸引力）のみが加わっている。従って、血小板産生装置１０は、吸引ポンプ１２の流量を制御することで巨核球に加わる力を制御することができる。保持部材１１の内部に引き込まれた巨核球Ａには、吸引力が加わっている箇所や保持部材１１の開口部１１ａとの接触部分にせん断応力が生じる。巨核球は、吸引力によってその細胞質の一部分が分離して血小板Ｃが産生される。 The megakaryocyte A held by the holding member 11 has a force in a direction in which it is drawn into the collection channel 7 due to a pressure difference between the platelet production chamber 5 and the collection channel 7 generated by the suction pump 12 which is an external force applying means. Will be added. That is, the megakaryocyte is pressed against the holding member 11 by a force drawn into the holding member 11. At this time, only the force (suction force) due to the pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the collection channel 7 is applied to the megakaryocyte A. Therefore, the platelet production apparatus 10 can control the force applied to the megakaryocyte by controlling the flow rate of the suction pump 12. In the megakaryocyte A drawn into the holding member 11, shear stress is generated at a portion where a suction force is applied and a contact portion with the opening 11 a of the holding member 11. In megakaryocytes, a portion of the cytoplasm is separated by suction force to produce platelets C.
　このように構成することで、血小板産生装置１０は、保持部材１１に保持されている巨核球に血小板産生室５の内部の圧力と回収流路７の内部の圧力との圧力差による力を加えることで血小板を産生させる。さらに、血小板産生装置１０は、産生された血小板のみが保持部材１１の開口部１１ａを通じて回収液に混入するので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置１０は、吸引ポンプ１２の流量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置１０は、効率よく血小板を産生することができる。 With this configuration, the platelet production device 10 applies a force due to a pressure difference between the pressure inside the platelet production chamber 5 and the pressure inside the collection channel 7 to the megakaryocyte held by the holding member 11. To produce platelets. Furthermore, since only the produced platelets are mixed in the collected liquid through the opening 11a of the holding member 11, the platelet producing apparatus 10 omits the step of separating megakaryocytes and platelets. Moreover, the platelet production apparatus 10 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the suction pump 12. Thereby, the platelet production apparatus 10 can produce platelets efficiently.
　以下では、図６と図７とを用いて、本発明に係る血小板産生装置の第三実施形態における血小板産生装置１３について説明する。 Hereinafter, the platelet production device 13 in the third embodiment of the platelet production device according to the present invention will be described with reference to FIGS. 6 and 7.
　図６に示すように、血小板産生装置１３は、巨核球が含まれる液体を循環させながら電荷間に発生するクーロン力により血小板を産生する循環電荷方式として構成されている。血小板産生装置１３は、貯留タンク２、循環流路３、供給ポンプ４、血小板産生室５、保持部材１４、回収タンク１５および電荷発生装置１６を具備している。 As shown in FIG. 6, the platelet production device 13 is configured as a circulating charge system that produces platelets by Coulomb force generated between charges while circulating a liquid containing megakaryocytes. The platelet production device 13 includes a storage tank 2, a circulation channel 3, a supply pump 4, a platelet production chamber 5, a holding member 14, a collection tank 15, and a charge generation device 16.
　血小板産生装置１３の培養液を循環させる構成は、前述の第一実施形態にかかる血小板産生装置１の構成と同一である。 The configuration for circulating the culture solution of the platelet production device 13 is the same as the configuration of the platelet production device 1 according to the first embodiment described above.
　図６と図７（ａ）とに示すように、保持手段である保持部材１４は、板状に形成されている。保持部材１４は、一側面と他側面とを連通するように開口部１４ａが形成されている。保持部材１４は、血小板産生室５にその空間を仕切るように設けられている。すなわち、保持部材１４の一側面は、血小板産生室５の内部に配置されて培養液に含まれている巨核球が保持部材１４の開口部１４ａ（一側）に接触するように構成されている。また、保持部材１４の他側面は、血小板産生室５の内部における回収流路７の一部を構成している。 As shown in FIG. 6 and FIG. 7A, the holding member 14 as a holding means is formed in a plate shape. The holding member 14 has an opening 14a so as to communicate one side and the other side. The holding member 14 is provided in the platelet production chamber 5 so as to partition the space. That is, one side surface of the holding member 14 is configured so that the megakaryocyte disposed in the platelet production chamber 5 and contained in the culture solution contacts the opening 14a (one side) of the holding member 14. . In addition, the other side surface of the holding member 14 constitutes a part of the collection flow path 7 in the platelet production chamber 5.
　具体的には、保持部材１４は、血小板産生室５の底部に配置されて、その一側面が血小板産生室５の底面を構成している。なお、本実施形態において、血小板産生室５内に設けられる保持部材１４の設置態様は、特に限定されるものではない。例えば、血小板産生室５の左右方向の途中部に配置され、血小板産生室５を左右に分割してもよい。また、開口部１４ａで保持する巨核球の数を増やすために、複数の保持部材１４によって血小板産生室５を複数の空間に分割するように構成して保持部材１４の表面積を増大させてもよい。 Specifically, the holding member 14 is disposed at the bottom of the platelet production chamber 5, and one side surface thereof constitutes the bottom surface of the platelet production chamber 5. In this embodiment, the installation mode of the holding member 14 provided in the platelet production chamber 5 is not particularly limited. For example, the platelet production chamber 5 may be arranged in the middle of the left-right direction of the platelet production chamber 5 and the platelet production chamber 5 may be divided into left and right. In addition, in order to increase the number of megakaryocytes held in the opening 14a, the platelet production chamber 5 may be divided into a plurality of spaces by the plurality of holding members 14 to increase the surface area of the holding member 14. .
　回収タンク１５は、産生された血小板を回収するものである。回収タンク１５は、血小板産生室５の下方に配置され、血小板産生室５の底部を覆うように接続されている。すなわち、回収タンク１５の上面は、保持部材１４の他側面から構成されている。これにより、回収タンク１５は、保持部材１４を介して血小板産生室５に接続されている。さらに、回収タンク１５は、保持部材１４の開口部１４ａを介して血小板産生室５に連通されている。 The collection tank 15 collects the produced platelets. The collection tank 15 is disposed below the platelet production chamber 5 and connected so as to cover the bottom of the platelet production chamber 5. That is, the upper surface of the collection tank 15 is configured from the other side surface of the holding member 14. Thereby, the collection tank 15 is connected to the platelet production chamber 5 via the holding member 14. Further, the collection tank 15 is communicated with the platelet production chamber 5 via the opening 14 a of the holding member 14.
　外力付与手段である電荷発生装置１６は、プラスの電荷を発生させるものである。電荷発生装置１６は、電極１６ａが回収タンク１５内の保持部材１４の近傍に配置されて、保持部材１４の他側面近傍にプラスの電荷を発生させるように構成されている。電荷発生装置１６は、任意の量のプラスの電荷を発生できるように構成されている。 The charge generator 16 which is an external force applying means generates positive charges. The charge generation device 16 is configured such that the electrode 16 a is disposed in the vicinity of the holding member 14 in the collection tank 15 and generates a positive charge in the vicinity of the other side surface of the holding member 14. The charge generator 16 is configured to generate an arbitrary amount of positive charges.
　このように構成される循環電荷方式の血小板産生装置１３は、貯留タンク２と血小板産生室５との間で培養液が循環するように構成されている。また、血小板産生装置１３は、電荷発生装置１６によってプラスの電荷を発生させることでマイナスの電荷を帯電している巨核球との間にクーロン力が生じるように構成されている。 The thus configured circulating charge type platelet production apparatus 13 is configured such that the culture medium circulates between the storage tank 2 and the platelet production chamber 5. In addition, the platelet production device 13 is configured such that a Coulomb force is generated between the platelet generator 13 and the megakaryocyte charged with a negative charge by generating a positive charge with the charge generator 16.
　次に、図７（ｂ）を用いて、血小板産生装置１３における血小板産生方法の態様について具体的に説明する。 Next, the embodiment of the platelet production method in the platelet production apparatus 13 will be specifically described with reference to FIG.
　図７（ｂ）に示すように、培養液とともに血小板産生室５に供給された巨核球と巨核球からすでに産生された血小板とは、一部の巨核球Ａと血小板とが保持部材１４に設けられている開口部１４ａの一側にその一部分がはまり込んで保持部材１４に保持される。一方、培養液とともに血小板産生室５に供給された血小板は、一部の血小板が保持部材１４の開口部１４ａに入り込む。 As shown in FIG. 7B, the megakaryocytes supplied to the platelet production chamber 5 together with the culture solution and the platelets already produced from the megakaryocytes are provided in the holding member 14 with some megakaryocytes A and platelets. A portion of the opening 14 a is fitted into one side of the opening 14 a and is held by the holding member 14. On the other hand, some of the platelets supplied to the platelet production chamber 5 together with the culture solution enter the opening 14 a of the holding member 14.
　巨核球は、常にマイナスの電荷を帯びている。つまり、保持部材１４に保持されている巨核球Ａには、外力付与手段である電荷発生装置１６によって回収タンク１５内で発生されたプラスの電荷との間で生じるクーロン力により回収タンク１５内に引き込まれる方向の力が加わる（細矢印参照）。すなわち、巨核球Ａは、回収タンク１５内に引き込まれる力によって保持部材１４に押し付けられている。この際、巨核球には、クーロン力により回収タンク１５内に引き込まれる方向の力のみが加わっている。従って、血小板産生装置１３は、電荷発生装置１６の電荷発生量を制御することで巨核球に加わる力を制御することができる。巨核球は、クーロン力が加わっている箇所や保持部材１４の開口部１４ａとの接触部分にせん断応力が生じる。 Megakaryocytes are always negatively charged. In other words, the megakaryocyte A held by the holding member 14 is placed in the recovery tank 15 by the Coulomb force generated between the positive charge generated in the recovery tank 15 by the charge generator 16 which is an external force applying means. A pulling force is applied (see thin arrows). That is, the megakaryocyte A is pressed against the holding member 14 by the force drawn into the collection tank 15. At this time, only the force in the direction of being drawn into the recovery tank 15 by the Coulomb force is applied to the megakaryocyte. Therefore, the platelet production device 13 can control the force applied to the megakaryocyte by controlling the charge generation amount of the charge generation device 16. In the megakaryocyte, a shear stress is generated at a portion where a Coulomb force is applied or a contact portion with the opening 14 a of the holding member 14.
　回収タンク１５に引き込まれた巨核球には、クーロン力によってせん断応力が発生している箇所を起点として巨核球を形成している細胞質の一部分がせん断される。巨核球は、クーロン力によってその細胞質の一部分が分離して血小板Ｃが産生される。産生された血小板Ｃは、回収タンク１５内の回収液中に混入して巨核球と分離された状態で回収される。 The part of the cytoplasm forming the megakaryocyte is sheared from the location where the shear stress is generated by the Coulomb force to the megakaryocyte drawn into the recovery tank 15. In megakaryocytes, a part of the cytoplasm is separated by Coulomb force and platelets C are produced. The produced platelet C is collected in a state separated from megakaryocytes by being mixed in the collection liquid in the collection tank 15.
　このように構成することで、血小板産生装置１３は、保持部材１４に保持されている巨核球に回収タンク１５内に引き込まれる方向のクーロン力を加えることで血小板を産生させる。さらに、血小板産生装置１３は、産生された血小板のみが保持部材１４の開口部１４ａを通じて回収されるので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置１３は、電荷発生装置１６の電荷発生量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置１３は、効率よく血小板を産生することができる。 With this configuration, the platelet production device 13 produces platelets by applying a Coulomb force in the direction of being drawn into the collection tank 15 to the megakaryocytes held by the holding member 14. Furthermore, since only the produced platelets are collected through the opening 14a of the holding member 14, the platelet producing device 13 omits the step of separating megakaryocytes and platelets. In addition, the platelet production device 13 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the charge generation amount of the charge generation device 16. Thereby, the platelet production apparatus 13 can produce platelets efficiently.
　以下では、図８を用いて、本発明に係る血小板産生装置の第四実施形態における血小板産生装置１７について説明する。 Hereinafter, the platelet production device 17 in the fourth embodiment of the platelet production device according to the present invention will be described with reference to FIG.
　図８に示すように、血小板産生装置１７は、巨核球が含まれる液体を拡販させながら回収液を循環させて血小板を産生する撹拌循環方式として構成されている。血小板産生装置１７は、貯留タンク２、供給流路１８、供給ポンプ４、血小板産生室１９、保持部材６、回収流路７、回収ポンプ８および回収タンク９を具備している。 As shown in FIG. 8, the platelet production device 17 is configured as an agitation circulation system that produces platelets by circulating the collected liquid while expanding the liquid containing megakaryocytes. The platelet production apparatus 17 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 6, a collection flow path 7, a collection pump 8, and a collection tank 9.
　供給流路１８は、培養液を供給するための流路である。供給流路１８は、非反応性ポリマー、生物親和性金属、ガラス等からなる配管から構成されている。供給流路１８は、供給ポンプ４を介して貯留タンク２と血小板産生室１９とを接続するように構成されている。すなわち、貯留タンク２内の培養液は、供給流路１８の途中部に設けられている供給ポンプ４によって供給流路１８を通じて血小板産生室１９に供給されるように構成されている。 The supply channel 18 is a channel for supplying a culture solution. The supply flow path 18 is comprised from piping which consists of a non-reactive polymer, a bioaffinity metal, glass, etc. The supply channel 18 is configured to connect the storage tank 2 and the platelet production chamber 19 via the supply pump 4. That is, the culture solution in the storage tank 2 is configured to be supplied to the platelet production chamber 19 through the supply channel 18 by the supply pump 4 provided in the middle of the supply channel 18.
　血小板産生室１９は、巨核球から血小板を産生させる空間を構成するものである。血小板産生室１９は、非反応性ポリマーまたは生物親和性金属等から形成される容器である。血小板産生室１９は、供給流路１８を通じて貯留タンク２に接続されている。つまり、血小板産生室１９は、供給流路１８を通じて貯留タンク２から培養液が供給されるように構成されている。血小板産生室１９には、供給された培養液を撹拌するための撹拌装置１９ａが設けられている。 The platelet production chamber 19 constitutes a space for producing platelets from megakaryocytes. The platelet production chamber 19 is a container formed from a non-reactive polymer or a biocompatible metal. The platelet production chamber 19 is connected to the storage tank 2 through the supply channel 18. That is, the platelet production chamber 19 is configured so that the culture solution is supplied from the storage tank 2 through the supply channel 18. The platelet production chamber 19 is provided with a stirring device 19a for stirring the supplied culture solution.
　このように構成される撹拌循環方式の血小板産生装置１７は、貯留タンク２内の培養液が供給ポンプ４によって供給流路１８を通じて血小板産生室１９に供給されるように構成されている。さらに、血小板産生装置１７は、血小板産生室１９内の培養液を撹拌装置１９ａによって撹拌できるように構成されている。また、血小板産生装置１７は、回収タンク９内の回収液が回収ポンプ８によって回収流路７を通じて保持部材６の内部に供給されるように構成されている。さらに、血小板産生装置１は、保持部材６の内部に供給された回収液が回収流路７を通じて回収タンク９に排出されるように構成されている。すなわち、血小板産生装置１７は、回収タンク９と保持部材６との間で回収液が循環するように構成されている。 The agitation and circulation type platelet production apparatus 17 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Further, the platelet production device 17 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. Further, the platelet production device 17 is configured such that the collected liquid in the collection tank 9 is supplied into the holding member 6 through the collection channel 7 by the collection pump 8. Further, the platelet production device 1 is configured such that the collected liquid supplied into the holding member 6 is discharged to the collection tank 9 through the collection channel 7. That is, the platelet production device 17 is configured such that the collected liquid circulates between the collection tank 9 and the holding member 6.
　次に、図３と図８とを用いて、血小板産生装置１７における血小板産生方法の態様について具体的に説明する。 Next, the embodiment of the platelet production method in the platelet production apparatus 17 will be specifically described with reference to FIGS.
　図３と図８に示すように、第一実施形態における血小板産生方法の態様と同様に、培養液とともに血小板産生室１９に供給された巨核球と巨核球からすでに産生された血小板とは、血小板産生室１９において一部の巨核球と血小板とが保持部材６に接触する。保持部材６に接触した一部の巨核球は、保持部材６に設けられている開口部６ａの一側にその一部分がはまり込んで保持部材６に保持される。保持されている巨核球には、回収ポンプ８が供給する回収液の流れによる力が加わっている。回収液の流れ方向に押された巨核球には、回収液の流れによる力が加わっている箇所や保持部材６の開口部６ａとの接触部分にせん断応力が生じる。巨核球は、回収液の流れによる力によってその細胞質の一部分が分離して血小板が産生される。 As shown in FIGS. 3 and 8, as in the platelet production method of the first embodiment, the megakaryocytes supplied to the platelet production chamber 19 together with the culture solution and the platelets already produced from the megakaryocytes are platelets. In the production chamber 19, some megakaryocytes and platelets contact the holding member 6. A part of the megakaryocytes in contact with the holding member 6 is held by the holding member 6 while part of the megakaryocyte is fitted into one side of the opening 6 a provided in the holding member 6. The held megakaryocyte is subjected to a force due to the flow of the recovery liquid supplied by the recovery pump 8. In the megakaryocyte pushed in the flow direction of the collected liquid, a shear stress is generated at a portion where a force due to the flow of the collected liquid is applied or a contact portion with the opening 6 a of the holding member 6. Megakaryocytes produce platelets by separating a part of their cytoplasm by the force of the flow of the collected liquid.
　培養液に含まれる巨核球のうち、保持部材６に接触しなかった巨核球や保持部材６に接触したが開口部６ａにはまり込まなかった巨核球は、血小板産生室１９の撹拌装置１９ａによって撹拌されている培養液の流れにより保持部材６に接触する方向に移動される（細矢印参照）。 Among megakaryocytes contained in the culture solution, megakaryocytes that did not contact the holding member 6 and megakaryocytes that contacted the holding member 6 but did not get stuck in the opening 6 a were stirred by the stirring device 19 a of the platelet production chamber 19. It is moved in the direction in contact with the holding member 6 by the flow of the culture broth (see thin arrows).
　このように構成することで、血小板産生装置１７は、保持部材６に保持されている巨核球に回収液の流れによる力を加えることで血小板を産生させる。さらに、血小板産生装置１７は、産生された血小板のみが回収液に混入して回収されるので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置１７は、回収ポンプ８の流量を変更することで巨核球に加わる力が血小板の産生に適切な値になるように設定される。これにより、血小板産生装置１７は、効率よく血小板を産生することができる。 With this configuration, the platelet production device 17 produces platelets by applying a force due to the flow of the recovered liquid to the megakaryocytes held by the holding member 6. Furthermore, since the platelet production apparatus 17 collects only the produced platelets and collects them in the collection liquid, the step of separating megakaryocytes and platelets is omitted. Further, the platelet production device 17 is set so that the force applied to the megakaryocyte becomes a value appropriate for the production of platelets by changing the flow rate of the collection pump 8. Thereby, the platelet production apparatus 17 can produce platelets efficiently.
　以下では、図９を用いて、本発明に係る血小板産生装置の第五実施形態における血小板産生装置２０について説明する。 Hereinafter, the platelet production apparatus 20 in the fifth embodiment of the platelet production apparatus according to the present invention will be described with reference to FIG.
　図９に示すように、血小板産生装置２０は、前述の第二実施形態にかかる血小板産生装置１０の培養液を供給ポンプ４で循環させる構成を撹拌装置１９ａで撹拌させる構成とし、吸引（圧力差）により血小板を産生させる撹拌吸引方式として構成されている。撹拌吸引方式の血小板産生装置２０は、貯留タンク２、供給流路１８、供給ポンプ４、血小板産生室１９、保持部材１１、回収流路７、吸引ポンプ１２および回収タンク９を具備している。 As shown in FIG. 9, the platelet production device 20 has a configuration in which the culture solution of the platelet production device 10 according to the second embodiment described above is circulated by the supply pump 4 and is agitated by the agitator 19 a, and suction (pressure difference) ) Is configured as a stirring and aspiration method for producing platelets. The stirring suction type platelet production apparatus 20 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 11, a collection flow path 7, a suction pump 12, and a collection tank 9.
　血小板産生装置２０の血小板産生室１９において培養液を撹拌させる構成は、前述の第四実施形態における血小板産生装置１７の構成と同一である。 The configuration of stirring the culture solution in the platelet production chamber 19 of the platelet production device 20 is the same as the configuration of the platelet production device 17 in the fourth embodiment described above.
　血小板産生装置２０の保持部材１１に保持された巨核球に吸引（圧力差）による力を加える構成は、前述の第二実施形態における血小板産生装置１０の構成と同一である。 The configuration for applying force by suction (pressure difference) to the megakaryocyte held by the holding member 11 of the platelet production device 20 is the same as the configuration of the platelet production device 10 in the second embodiment described above.
　このように構成される撹拌吸引方式の血小板産生装置２０は、貯留タンク２内の培養液が供給ポンプ４によって供給流路１８を通じて血小板産生室１９に供給されるように構成されている。さらに、血小板産生装置２０は、血小板産生室１９内の培養液を撹拌装置１９ａで撹拌できるように構成されている。また、血小板産生装置２０は、吸引ポンプ１２によって保持部材１１の内部の圧力と保持部材１１の外部である血小板産生室１９の内部の圧力との間に圧力差が生じるように構成されている。 The agitation and suction type platelet production apparatus 20 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Further, the platelet production device 20 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. In addition, the platelet production device 20 is configured such that a pressure difference is generated between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 19 that is outside the holding member 11 by the suction pump 12.
　このように構成することで、血小板産生装置２０は、保持部材１１に保持されている巨核球に血小板産生室１９と回収流路７との圧力差による力を加えることで血小板を産生させる。さらに、血小板産生装置２０は、産生された血小板のみが保持部材１１の開口部１１ａを通じて回収されるので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置２０は、吸引ポンプ１２の流量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置２０は、効率よく血小板を産生することができる。 With this configuration, the platelet production apparatus 20 produces platelets by applying a force due to a pressure difference between the platelet production chamber 19 and the collection flow path 7 to the megakaryocytes held by the holding member 11. Furthermore, since only the produced platelets are collected through the opening 11a of the holding member 11, the platelet producing apparatus 20 omits the step of separating megakaryocytes and platelets. Moreover, the platelet production apparatus 20 is set so that force appropriate for the production of platelets is applied to the megakaryocyte by controlling the flow rate of the suction pump 12. Thereby, the platelet production apparatus 20 can produce platelets efficiently.
　以下では、図１０を用いて、本発明に係る血小板産生装置の第六実施形態における血小板産生装置２１について説明する。 Hereinafter, the platelet production apparatus 21 in the sixth embodiment of the platelet production apparatus according to the present invention will be described with reference to FIG.
　図１０に示すように、血小板産生装置２１は、前述の第三実施形態にかかる血小板産生装置１３の培養液を供給ポンプ４で循環させる構成を撹拌装置１９ａで撹拌させる構成とし、電荷間に発生するクーロン力により血小板を産生する撹拌電荷方式として構成されている。撹拌電荷方式の血小板産生装置２１は、貯留タンク２、供給流路１８、供給ポンプ４、血小板産生室１９、保持部材１４、回収タンク１５および電荷発生装置１６を具備している。 As shown in FIG. 10, the platelet production device 21 has a configuration in which the culture solution of the platelet production device 13 according to the third embodiment described above is circulated by the supply pump 4 and is stirred by the stirring device 19a, and is generated between charges. It is configured as a stirring charge system that produces platelets by Coulomb force. The stirred charge type platelet production device 21 includes a storage tank 2, a supply flow path 18, a supply pump 4, a platelet production chamber 19, a holding member 14, a collection tank 15, and a charge generation device 16.
　血小板産生装置２１の血小板産生室１９において培養液を撹拌させる構成は、前述の第四実施形態における血小板産生装置１７の構成と同一である。 The configuration of stirring the culture solution in the platelet production chamber 19 of the platelet production device 21 is the same as the configuration of the platelet production device 17 in the fourth embodiment described above.
　血小板産生装置２１の保持部材１１に保持された巨核球に帯電した電荷を利用してクーロン力を加える構成は、前述の第三実施形態における血小板産生装置１３の構成と同一である。 The configuration for applying the Coulomb force using the electric charge charged to the megakaryocytes held by the holding member 11 of the platelet production device 21 is the same as the configuration of the platelet production device 13 in the third embodiment described above.
　このように構成される撹拌吸引方式の血小板産生装置２１は、貯留タンク２内の培養液が供給ポンプ４によって供給流路１８を通じて血小板産生室１９に供給されるように構成されている。さらに、血小板産生装置２１は、血小板産生室１９内の培養液を撹拌装置１９ａによって撹拌できるように構成されている。また、血小板産生装置２１は、電荷発生装置１６によってプラスの電荷を発生させることでマイナスの電荷を帯電している巨核球との間にクーロン力が生じるように構成されている。 The agitation and suction type platelet production apparatus 21 configured as described above is configured such that the culture solution in the storage tank 2 is supplied to the platelet production chamber 19 through the supply flow path 18 by the supply pump 4. Furthermore, the platelet production device 21 is configured so that the culture solution in the platelet production chamber 19 can be stirred by the stirring device 19a. Further, the platelet production device 21 is configured such that a Coulomb force is generated between the megakaryocyte charged with a negative charge by generating a positive charge with the charge generation device 16.
　このように構成することで、血小板産生装置２１は、保持部材１４に保持されている巨核球に回収タンク１５内に引き込まれる方向のクーロン力を加えることで血小板を産生させる。さらに、血小板産生装置２１は、産生された血小板のみが保持部材１４の開口部１１ａを通じて回収されるので巨核球と血小板とを分離する工程が省かれる。また、血小板産生装置２１は、電荷発生装置１６の電荷発生量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置２１は、効率よく血小板を産生することができる。 With this configuration, the platelet production device 21 produces platelets by applying Coulomb force in the direction of being drawn into the collection tank 15 to the megakaryocytes held by the holding member 14. Furthermore, since only the produced platelets are collected through the opening 11a of the holding member 14, the platelet producing apparatus 21 omits the step of separating megakaryocytes and platelets. In addition, the platelet production device 21 is set so that a force appropriate for the production of platelets is applied to the megakaryocyte by controlling the amount of charge generated by the charge generation device 16. Thereby, the platelet production apparatus 21 can produce platelets efficiently.
　なお、前述の第一実施形態から第六実施形態に係る血小板産生装置において、血小板の産生時に血小板と合わせて回収される培養液の不足分を補うため、血小板産生装置１と血小板産生装置１０とにおける血小板産生室５と回収タンク９との間、血小板産生装置１３における血小板産生室５と回収タンク１５との間、血小板産生装置１７と血小板産生装置２０とにおける血小板産生室１９と回収タンク９との間、および血小板産生装置２１における血小板産生室１９と回収タンク１５との間で回収液を循環させる構成や生理食塩水等を所定のタイミングで貯留タンク２や血小板産生室５・１９に供給する構成としてもよい。 In addition, in the platelet production device according to the first to sixth embodiments, the platelet production device 1 and the platelet production device 10 are used to compensate for the shortage of the culture solution collected together with the platelets at the time of platelet production. Between the platelet production chamber 5 and the collection tank 9, between the platelet production chamber 5 and the collection tank 15 in the platelet production device 13, and between the platelet production chamber 19 and the collection tank 9 in the platelet production device 17 and the platelet production device 20. Between the platelet production chamber 19 and the collection tank 15 in the platelet production apparatus 21 and the physiological saline and the like are supplied to the storage tank 2 and the platelet production chambers 5 and 19 at a predetermined timing. It is good also as a structure.
　また、前述の第一実施形態から第六実施形態における巨核球に力を加える態様を組み合わせて複数の力を巨核球に加えてもよい。例えば、前述の第一実施形態から第三実施形態に係る血小板産生装置１、血小板産生装置１０および血小板産生装置１３において、供給ポンプ４を外力付与手段として、循環流路３に供給されている培養液の流れによって巨核球に力を加えてもよい。血小板産生装置１、血小板産生装置１０および血小板産生装置１３は、更に供給ポンプ４の流量を制御することによって血小板の産生に適切な力が巨核球に加わるように設定される。これにより、血小板産生装置１０は、効率よく血小板を産生することができる。 Further, a plurality of forces may be applied to the megakaryocyte by combining the modes for applying the force to the megakaryocyte in the first to sixth embodiments described above. For example, in the platelet production apparatus 1, the platelet production apparatus 10, and the platelet production apparatus 13 according to the first to third embodiments described above, the culture that is supplied to the circulation flow path 3 using the supply pump 4 as an external force applying means. A force may be applied to the megakaryocyte by the flow of the liquid. The platelet production apparatus 1, the platelet production apparatus 10, and the platelet production apparatus 13 are set so that a force appropriate for the production of platelets is applied to the megakaryocytes by further controlling the flow rate of the supply pump 4. Thereby, the platelet production apparatus 10 can produce platelets efficiently.
　また、前述の第一実施形態に係る血小板産生装置１と第四実施形態に係る血小板産生装置１７との回収流路７の構成において、回収ポンプ８を保持部材６の下流側に設けるとともに、保持部材６の上流側の回収流路７に絞りを設けることで保持部材１１の内部の圧力を低下させて、保持部材１１の内部の圧力と血小板産生室１０の内部の圧力とに圧力差を生じさせてもよい。これにより、保持部材６に保持されている巨核球には、開口部６ａにはまり込んでいる部分に回収流路７内を流れている回収液からその流れ方向に押す力が加わるとともに、血小板産生室５または血小板産生室１９と回収流路７との圧力差による回収流路７内に引き込まれる方向の力が加わる。この際、巨核球には、回収ポンプ８が供給する回収液の流れによる力と吸引ポンプ１２の吸引力とが加わっている。従って、血小板産生装置２１は、供給ポンプ４と吸引ポンプ１２との流量を制御することで巨核球に加わる力を制御することができる。 Further, in the configuration of the recovery flow path 7 of the platelet production device 1 according to the first embodiment and the platelet production device 17 according to the fourth embodiment, the collection pump 8 is provided on the downstream side of the holding member 6 and held. By providing a restriction in the collection flow path 7 on the upstream side of the member 6, the pressure inside the holding member 11 is lowered, and a pressure difference is generated between the pressure inside the holding member 11 and the pressure inside the platelet production chamber 10. You may let them. As a result, the megakaryocyte held by the holding member 6 is applied with a force that pushes in the flow direction from the collected liquid flowing in the collection flow path 7 to the portion of the megakaryocyte that is stuck in the opening 6a, and also produces platelets. A force in the direction of being drawn into the collection channel 7 due to a pressure difference between the chamber 5 or the platelet production chamber 19 and the collection channel 7 is applied. At this time, the megakaryocyte is applied with the force of the flow of the recovery liquid supplied by the recovery pump 8 and the suction force of the suction pump 12. Therefore, the platelet production device 21 can control the force applied to the megakaryocyte by controlling the flow rates of the supply pump 4 and the suction pump 12.
　加えて、巨核球に帯電しているマイナスの電荷と電荷発生装置１６によって発生されたプラスの電荷との間で生じるクーロン力により保持部材６の内部に引き込まれる方向の力を生じさせてもよい。従って、血小板産生装置２１は、供給ポンプ４と回収ポンプ８との流量および電荷発生装置１６の電荷発生量を制御することで巨核球に加わる力を制御することができる。 In addition, a force in a direction of being drawn into the holding member 6 may be generated by a Coulomb force generated between a negative charge charged in the megakaryocyte and a positive charge generated by the charge generation device 16. . Therefore, the platelet production device 21 can control the force applied to the megakaryocyte by controlling the flow rates of the supply pump 4 and the recovery pump 8 and the charge generation amount of the charge generation device 16.
　また、第一実施形態から第六実施形態における保持部材６、保持部材１１および保持部材１４は、有核細胞である巨核球の捕捉能が高い有核細胞捕捉材によって形成して巨核球を保持する構成でもよい。捕捉材として、ポリエチレン、ポリプリロピレン、ポリスチレン、アクリル樹脂、ナイロン、ポリエステル、ポリカーボネート、ポリアクリルアミド、ポリウレタン等の合成高分子、アガロース、セルロース、酢酸セルロース、キチン、キトサン、アルギン酸塩等の天然高分子、ハイドロキシアパタイト、ガラス、アルミナ、チタニア等の無機材料、ステンレス、チタン、アルミニウム等の金属があげられる。また、これらの捕捉材はこのままでも用いることができるが、血小板通過性を高める、あるいは細胞の選択的捕捉を行う等の必要に応じ、表面改質を施したものでもよい。例えば、血小板通過性を高めるにはＷＯ８７／０５８１２公報で提案されている非イオン性親水基と塩基性含窒素官能基を有するポリマーのコートによる方法等があげられる。 In addition, the holding member 6, the holding member 11, and the holding member 14 in the first to sixth embodiments are formed of a nucleated cell-capturing material having a high ability to capture megakaryocytes that are nucleated cells to hold the megakaryocytes. The structure to do may be sufficient. As capture materials, polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane and other synthetic polymers, agarose, cellulose, cellulose acetate, chitin, chitosan, alginates and other natural polymers, Examples thereof include inorganic materials such as hydroxyapatite, glass, alumina and titania, and metals such as stainless steel, titanium and aluminum. In addition, these capture materials can be used as they are, but they may be subjected to surface modification as necessary, for example, to increase platelet permeability or to selectively capture cells. For example, in order to increase the platelet permeability, there can be mentioned a method of coating a polymer having a nonionic hydrophilic group and a basic nitrogen-containing functional group proposed in WO 87/05812.
　図１１に示すように、前述の第一実施形態から第六実施形態にかかる血小板産生装置は、ｉＰＳ細胞から誘導された巨核球前駆細胞を所定の条件で培養し、成熟した巨核球を製造する培養装置２３、培養液中の巨核球量を検出するための液中パーティクルカウンター等から構成される検出装置２４、回収液から巨核球の核やＤＮＡを除去するごみ分離装置２５および回収した血小板を濃縮する濃縮装置２６を具備する血小板産生システム２２として構成してもよい。このように構成することで、ｉＰＳ細胞から誘導された巨核球前駆細胞から血小板製剤を連続的に製造することができる。 As shown in FIG. 11, the platelet production apparatus according to the first to sixth embodiments described above cultivates megakaryocyte progenitor cells derived from iPS cells under predetermined conditions to produce mature megakaryocytes. A culture device 23, a detection device 24 comprising a submerged particle counter or the like for detecting the amount of megakaryocytes in the culture solution, a dust separation device 25 for removing megakaryocyte nuclei and DNA from the recovery solution, and recovered platelets You may comprise as the platelet production system 22 provided with the concentration apparatus 26 which concentrates. By comprising in this way, a platelet formulation can be continuously manufactured from the megakaryocyte precursor cell induced | guided | derived from the iPS cell.
　１　血小板産生装置
　４　供給ポンプ
　５　血小板産生室
　６　保持部材
　８　回収ポンプ 1 Platelet Production Device 4 Supply Pump 5 Platelet Production Chamber 6 Holding Member 8 Collection Pump

Claims (6)

  1. 　巨核球が含まれる液体を貯留する容器と、
    　容器に巨核球が含まれる液体を供給する供給手段と、
    　容器の一部にもうけられ、巨核球を保持する保持手段と、
    　前記保持手段により保持された巨核球に外力を加えて血小板を産生させる外力付与手段と、を具備する血小板産生装置。     A container for storing a liquid containing megakaryocytes,
    Supply means for supplying a liquid containing megakaryocytes in a container;
    A holding means provided in a part of the container and holding a megakaryocyte;
    An external force applying means for applying an external force to the megakaryocytes held by the holding means to produce platelets.
  2. 　前記保持手段に前記血小板が通過できて前記巨核球が通過できない大きさの開口部が形成され、開口部の一側に巨核球の一部分がはまり込むことで巨核球を保持する請求項１に記載の血小板産生装置。 2. The megakaryocyte is held by forming an opening having a size that allows the platelets to pass through the holding means but not allow the megakaryocyte to pass through, and a part of the megakaryocyte fits into one side of the opening. Platelet production equipment.
  3. 　前記外力付与手段が前記開口部の他側から流体の流れにより前記巨核球に外力を加える請求項１または請求項２に記載の血小板産生装置。 3. The platelet production apparatus according to claim 1, wherein the external force applying means applies an external force to the megakaryocyte by a fluid flow from the other side of the opening.
  4. 　前記外力付与手段が前記開口部の一側と他側との間に圧力差を生じさせることにより前記巨核球に外力を加える請求項１から請求項３のいずれか一項に記載の血小板産生装置。 The platelet production apparatus according to any one of claims 1 to 3, wherein the external force applying means applies an external force to the megakaryocyte by generating a pressure difference between one side and the other side of the opening. .
  5. 　前記外力付与手段が巨核球に帯電している電荷と異なる電荷を前記開口部の他側に生じさせることにより前記巨核球に外力を加える請求項１から請求項４のいずれか一項に記載の血小板産生装置。 The said external force provision means applies external force to the said megakaryocyte by producing the electric charge different from the electric charge which is charged in the megakaryocyte on the other side of the said opening part. Platelet production device.
  6. 　巨核球を保持し、外部から巨核球に力を加えて、巨核球から血小板を産生させる血小板産生方法。 A platelet production method that holds megakaryocytes and applies platelets from the outside to produce platelets from megakaryocytes.
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