JP2014155471A - Passage device for platelet production and platelet production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a passage device for platelet production which produces a lot of platelets effectively from megakaryocytes in vitro and to provide a platelet production method.SOLUTION: A passage device for platelet production is equipped with: a porous thin film (10) in which multiple micropores (13) are formed in a controlled form and arrangement, and megakaryocytes (M) are positioned on one side of a surface (11); a culture chamber (20) equipped in one side of the porous thin film, and in which the megakaryocytes are cultured; and a micro passage (30) equipped in the other side of the porous thin film, and in which a culture medium (S) flows while the megakaryocytes are cultured in the culture chamber.

Description

  The present invention relates to a platelet production channel device and a platelet production method, and more particularly to a channel device for mass-producing platelets from megakaryocytes outside the body.

  Platelets are produced in the body when hematopoietic progenitor cells differentiate into megakaryocytes in the bone marrow, and the cytoplasm of the megakaryocytes is fragmented and released into the blood. In recent years, it has also been reported that platelets are produced from megakaryocytes outside the body using iPS cells (induced Pluripotent Stem cells).

  However, since platelets cannot be stored frozen, they can only be stored for a few days after blood containing the platelets is collected or produced using megakaryocytes derived from iPS cells. On the other hand, hematopoietic progenitor cells can be stored frozen.

  Therefore, in Patent Document 1, a composite membrane produced by laminating a support porous membrane such as a nonwoven fabric and a porous thin film of an organic polymer is immersed in a culture solution, and two of the regions partitioned by the porous thin membrane are used. The platelet precursor cells are arranged in the region on the supporting porous membrane side, and a shear flow is generated in the region on the porous thin film side using a stirrer to apply shear stress to the porous thin film, and the platelet precursor cells Methods for producing more platelets have been proposed.

JP 2009-297023 A

  However, in the method described in Patent Document 1, for example, shear stress is generated by a rotating flow, so that a distribution occurs in the flow rate of the culture solution within the surface of the porous thin film, and uniform shear stress is applied to the entire surface. There is a problem that the platelet progenitor cells cannot be directly controlled because the platelet progenitor cells are arranged on a support porous membrane such as a nonwoven fabric.

  The present invention has been made in view of the above problems, and an object thereof is to provide a platelet production channel device and a platelet production method for effectively mass-producing platelets from megakaryocytes outside the body.

  A platelet production channel device according to an embodiment of the present invention for solving the above problems is a porous structure in which a plurality of micropores are formed in a controlled shape and arrangement, and a megakaryocyte is arranged on the surface of one side thereof. A thin film, a culture chamber provided on the one side of the porous thin film and in which the megakaryocytes are cultured, and a culture solution provided on the other side of the porous thin film and while the megakaryocytes are cultured in the culture chamber. And a micro flow path through which the gas flows. ADVANTAGE OF THE INVENTION According to this invention, the platelet production flow-path apparatus which effectively produces large quantities of platelets from megakaryocytes outside the body can be provided.

  Moreover, the hole diameter of each of the plurality of minute holes may be 2 to 15 μm. The average pore diameter of the plurality of micropores may be 2 to 15 μm. The plurality of minute holes may be formed with a hole diameter processing accuracy of ± 10% or less. Moreover, the thickness of the porous thin film may be 5 to 40 μm. Further, the aperture ratio of the plurality of micropores may be 4 to 45%. Moreover, the said microchannel is good also as being formed so that the said culture solution can be flowed with the flow rate of 1-100 mm / sec.

  The platelet production channel device may further include a temperature control unit that controls the temperature in the vicinity of the porous thin film. In this case, the temperature control unit may control the temperature to 30 to 40 ° C.

  In order to solve the above problems, a platelet production method according to an embodiment of the present invention includes a porous thin film in which a plurality of micropores are formed in a controlled shape and arrangement, and a culture provided on one side of the porous thin film Preparing a platelet production channel device comprising a chamber and a microchannel provided on the other side of the porous thin film, and arranging megakaryocytes on the surface of the one side of the porous thin film in the culture chamber , Culturing the megakaryocytes in the culture chamber while flowing a culture solution in the microchannel, producing platelets, and being released into the culture solution in the microchannel through the micropores Collecting the platelets. According to the present invention, it is possible to provide a method for producing platelets that effectively mass-produces platelets from megakaryocytes outside the body.

  In the platelet production method, the culture solution may be flowed through the microchannel at a flow rate of 1 to 100 mm / second.

  ADVANTAGE OF THE INVENTION According to this invention, the platelet production flow-path apparatus and platelet production method which produce large quantities of platelets effectively from megakaryocytes outside the body can be provided.

It is explanatory drawing which shows roughly a mode that platelets are produced from a megakaryocyte using the basic structure of an example of the platelet production flow-path apparatus which concerns on one Embodiment of this invention, and the said apparatus. It is explanatory drawing which shows the cross section of the platelet production flow-path apparatus used in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows the platelet production flow-path apparatus used in the Example which concerns on one Embodiment of this invention by planar view. It is explanatory drawing which shows the porous thin film formed in the Example which concerns on one Embodiment of this invention by planar view. It is explanatory drawing which shows an example of the arrangement pattern of the micropore in the porous thin film which concerns on one Embodiment of this invention. It is explanatory drawing which shows the other example of the arrangement pattern of the micropore in the porous thin film which concerns on one Embodiment of this invention. It is explanatory drawing which shows one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows another one of the some process included in formation of the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows an example of the result of having observed the megakaryocyte arrange | positioned at the porous thin film in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows an example of the result of having evaluated the production of the platelet from a megakaryocyte in the Example which concerns on one Embodiment of this invention. It is explanatory drawing which shows roughly the basic structure of the other example of the platelet production channel apparatus which concerns on one Embodiment of this invention.

  Hereinafter, an embodiment of the present invention will be described. Note that the present invention is not limited to this embodiment.

  A platelet production channel device according to this embodiment (hereinafter referred to as “the present device”) is a device that produces platelets P from megakaryocytes M outside the body. The platelet production method according to this embodiment (hereinafter referred to as “the present method”) is a method of producing platelets P from megakaryocytes M outside the body.

  FIG. 1 schematically shows the basic structure of an example of the present apparatus 1 and how platelets P are produced from megakaryocytes M using the present apparatus 1 according to the present method. As shown in FIG. 1, the apparatus 1 includes a porous thin film 10 in which a plurality of micropores 13 are formed in a controlled shape and arrangement, and a megakaryocyte M is arranged on a surface 11 on one side thereof. The culture chamber 20 for culturing the megakaryocyte M and the other side of the porous thin film 10 are provided on the one side of the membrane, and the megakaryocyte M is cultured in the culture chamber 20 to produce platelets P. And a micro flow channel 30 through which the culture solution S flows.

  The present method also includes a porous thin film 10 in which a plurality of micropores 13 are formed in a controlled shape and arrangement, a culture chamber 20 provided on one side of the porous thin film 10, and the other side of the porous thin film 10. A platelet production flow path device (this apparatus 1) provided with a micro flow path 30 provided on the surface of the porous thin film 10 in the culture chamber 20 and the megakaryocytes M on the one surface 11 of the porous thin film 10. Arranging, culturing the megakaryocyte M in the culture chamber 20 while flowing the culture medium S through the microchannel 30 to produce platelets P, and the microchannel 30 via the micropores 13. Recovering the platelets P released into the culture medium S.

  The porous thin film 10 is particularly limited as long as it has a plurality of micropores 13 formed in a controlled shape and arrangement and has a surface 11 on which the megakaryocyte M is arranged when the megakaryocyte M is cultured in the apparatus 1. I can't.

  For example, the porous thin film 10 may be formed by fine processing of a substrate. The microfabrication is, for example, a MEMS (Micro Electro Mechanical Systems) processing technique, and more specifically, for example, photolithography.

  The porous thin film 10 may be formed by, for example, cutting a part of the substrate and reducing the thickness by fine processing such as etching. In this case, the present apparatus 1 includes a porous thin film 10 formed by cutting a part of the substrate by microfabrication and reducing the thickness thereof, and another substrate that is thicker than the porous thin film 10 and surrounds the porous thin film 10. It is good also as providing the board | substrate part which has a frame part comprised from one part (after-mentioned).

  The material which comprises the porous thin film 10 is not specifically limited, An inorganic material and / or an organic material can be used. The porous thin film 10 made of an inorganic material is preferable in that it has excellent heat resistance. The porous thin film 10 made of an inorganic material is formed, for example, by fine processing of a substrate made of an inorganic material (for example, a silicon wafer).

  The porous thin film 10 may have a flat surface 11 on one side where the megakaryocytes M are arranged and a flat surface 12 on the other side. Further, the surface 11 on one side and the surface 12 on the other side may be formed in parallel.

  The thickness of the porous thin film 10 is such that the porous thin film 10 has a mechanical strength that can withstand the arrangement and culture of the megakaryocytes M on one surface 11 thereof, and the micropores of the platelets P produced from the megakaryocytes M Although it will not be restricted especially if it is the range which does not prevent the discharge | release to the microchannel 30 through 13 notably, For example, it may be 5-40 micrometers, may be 5-30 micrometers, and 5-20 micrometers. It is good also as being.

  For example, when the thickness of the porous thin film 10 is 5 to 20 μm, the porous thin film 10 has a structure similar to the blood vessel wall (thickness of about 10 μm) in the in vivo environment where platelets P are produced from the megakaryocytes M. It is preferable.

  The plurality of micro holes 13 are not particularly limited as long as they are formed in a controlled shape and arrangement. The plurality of microholes 13 formed in the controlled shape and arrangement may be, for example, a plurality of microholes 13 formed by microfabrication of the substrate. The fine processing is, for example, a MEMS processing technique, and more specifically, for example, photolithography.

  The micropore 13 is a hole penetrating from the surface 11 on one side to the surface 12 on the other side of the porous thin film 10. Moreover, the micropore 13 is formed in the shape and size which cannot pass the megakaryocyte M but platelet P can pass.

  The pore diameter of each of the plurality of micropores 13 (the diameter of each micropore 13) is not particularly limited as long as the megakaryocyte M cannot pass through the micropore 13 and the platelet P can pass through. It may be 15 μm, may be 2 to 10 μm, or may be 2 to 5 μm.

  For example, when the pore diameter of each micropore 13 is 2 to 5 μm, it is possible not only to prevent mature megakaryocytes from passing through the micropores 13 but also immature megakaryocytes pass through the micropores 13. This can also be reliably prevented.

  The average pore diameter of the plurality of micropores 13 is not particularly limited as long as the megakaryocytes M cannot pass through the micropores 13 and the platelets P can pass therethrough, but may be 2 to 15 μm, for example. It is good also as being 10 micrometers, and good also as being 2-5 micrometers.

  The plurality of minute holes 13 may be formed with, for example, a hole diameter processing accuracy of ± 10% or less. That is, the plurality of microholes 13 formed in a controlled shape and arrangement may be a plurality of microholes 13 formed with a hole diameter machining accuracy of ± 10% or less.

  Further, the plurality of microholes 13 formed in a controlled shape and arrangement are, for example, a plurality of microholes 13 having an average hole diameter of 2 to 15 μm and a hole diameter machining accuracy of ± 10% or less. Also good. In this case, the average pore diameter of the plurality of micropores 13 may be 2 to 10 μm, or 2 to 5 μm. For example, when a plurality of micropores 13 having an average pore diameter of 10 μm are formed in the porous thin film 10 with a pore diameter processing accuracy of ± 10%, the pore diameter of the plurality of micropores 13 is in the range of 9 to 11 μm. .

  The aperture ratio of the plurality of micropores 13 is such that the porous thin film 10 has a mechanical strength that can withstand the arrangement and culture of the megakaryocytes M on one surface 11 thereof, and the platelets P produced from the megakaryocytes M Although it will not be restricted especially if it is a range which does not prevent the discharge | release to the microchannel 30 through the micropore 13 notably, For example, it may be 4 to 45% and may be 4 to 35%. It may be 4 to 25%.

  The culture chamber 20 is not particularly limited as long as it is provided on the surface 11 side where the megakaryocyte M of the porous thin film 10 is arranged, and the megakaryocyte M is cultured in the inside thereof. The culture chamber 20 is a chamber that is sealed except that it communicates with the microchannel 30 via the micropores 13 of the porous thin film 10 while the megakaryocytes M are cultured therein. In addition, the surface 11 on which the megakaryocyte M of the porous thin film 10 is disposed may constitute an inner wall surface of the culture chamber 20 on the microchannel 30 side.

  In culturing the megakaryocyte M, the culture chamber 20 contains a culture solution S containing the megakaryocyte M. That is, the megakaryocytes M arranged on the surface 11 of the porous thin film 10 are cultured in the culture solution S accommodated in the culture chamber 20.

  The micro flow path 30 is not particularly limited as long as it is provided on the opposite side of the surface 11 on which the megakaryocytes M of the porous thin film 10 are disposed and the culture solution S flows. In the microchannel 30, the culture solution S flows while in contact with the surface 12 opposite to the surface 11 on which the megakaryocytes M of the porous thin film 10 are disposed.

  The microchannel 30 is linearly at least from the upstream end to the downstream end of the porous thin film 10 in the direction in which the culture solution S flows (the direction indicated by the arrow X shown in FIG. 1) (hereinafter referred to as “flow direction X”). It may be formed. In this case, the length of the microchannel 30 in the flow direction X is longer than that of the porous thin film 10. For this reason, the culture solution S in the microchannel 30 flows while contacting from the upstream end to the downstream end of the surface 12 of the porous thin film 10 in the flow direction X.

  Further, the microchannel 30 is a direction along the surface 12 of the porous thin film 10 and is orthogonal to the flowing direction (hereinafter referred to as “width direction”) from one end of the porous thin film 10 to the other end. It is good also as having been formed. In this case, the length in the width direction of the microchannel 30 (the width of the microchannel 30) is longer than that of the porous thin film 10. For this reason, the culture solution S in the microchannel 30 flows while contacting from one end to the other end of the surface 12 of the porous thin film 10 in the width direction.

  As described above, the microchannel 30 is formed linearly over the entire surface 12 of the porous thin film 10, so that the culture solution S is in contact with the entire surface 12 in the microchannel 30. As a result, uniform shear stress can be applied to the entire surface 12.

  The microchannel 30 may be formed so that the culture solution S can flow at a flow rate of 1 to 100 mm / second. That is, the microfluidic channel 30 contains the culture medium S within a range of 1 mm / second (flow rate equivalent to the blood flow rate in the capillary of the living body) to 100 mm / second (flow rate equivalent to the blood flow rate in the vein of the living body). It is formed in a shape and size that can flow. In this case, the culture fluid S flow simulating the blood flow in the blood vessel in the living body can be formed in the microchannel 30.

  In this method, the megakaryocyte M is arranged on the surface 11 on one side of the porous thin film 10 in the culture chamber 20 of the apparatus 1 as described above. The arrangement of the megakaryocytes M is performed, for example, by putting a culture solution S containing the megakaryocytes M into the culture chamber 20 and allowing the megakaryocytes M to settle on the surface 11 of the porous thin film 10. Also, the arrangement of the megakaryocytes M is, for example, by putting a culture solution S containing the precursor cells of the megakaryocytes M into the culture chamber 20, allowing the precursor cells to settle on the surface 11 of the porous thin film 10, and further It may be performed by culturing the precursor cells in the chamber 20 and differentiating them into the megakaryocytes M.

  The origin of the megakaryocyte M is not particularly limited, and may be a human megakaryocyte, or may be a megakaryocyte of an animal other than a human. The megakaryocyte M may be a megakaryocyte derived from blood collected from a living body, or may be a megakaryocyte derived from an iPS cell or ES cell, and is a stocked megakaryocyte. It is good also as it being a megakaryocyte derived from the established progenitor cell.

  Next, in this method, the megakaryocyte M is cultured in the culture chamber 20 while flowing the culture medium S through the microchannel 30 to produce platelets P. Specifically, for example, as shown in FIG. 1, one side of the porous thin film 10 is sealed in the sealed culture chamber 20 except that it communicates with the microchannel 30 via the plurality of micropores 13 of the porous thin film 10. By flowing the culture solution S from the upstream end to the downstream end of the microchannel 30 in contact with the surface 12 on the other side of the porous thin film 10 with the megakaryocytes M arranged on the surface 11, the culture chamber While culturing the megakaryocyte M within 20, the platelet P is produced from the megakaryocyte M.

  Here, by flowing the culture medium S through the microchannel 30, the meganuclear disposed on the surface 11 of the porous thin film 10 opposite to the microchannel 30 through the plurality of micropores 13 of the porous thin film 10. Share stress that promotes the production of platelets P can be applied to the sphere M.

  In particular, as described above, when the porous thin film 10 has the flat surfaces 11 and 12 and the microchannel 30 is formed linearly along the entire porous thin film 10, Uniform shear stress can be applied to the megakaryocytes M dispersed throughout.

  The flow rate of the culture medium S in the microchannel 30 is not particularly limited as long as it can give the megakaryocyte M a shear stress that promotes the production of platelets P and does not significantly disturb the recovery of the platelets P. However, for example, the culture solution S may be flowed through the microchannel 30 at a flow rate of 1 to 100 mm / second.

  In this case, the flow rate of the culture solution S in the microchannel 30 is set to a range from 1 mm / second equivalent to the blood flow rate in the capillary of the living body to 100 mm / second equivalent to the blood flow rate in the vein of the living body. Thus, the culture fluid S flow simulating the blood flow in the blood vessel in the living body can be formed in the microchannel 30, and the production of platelets P from the megakaryocytes M can be effectively promoted.

  The flow rate of the culture medium S in the microchannel 30 is determined by the concentration of the platelet P in the culture medium S calculated based on the production rate of the platelet P in the apparatus 1 and the flow rate of the culture medium S. It is good also as determining so that it may become the range which does not have trouble in the collection of the platelet P concerned.

  In this method, platelets P are produced from the megakaryocytes M by culturing the megakaryocytes M on the surface 11 of the porous thin film 10. That is, the megakaryocyte M deforms a part of its cytoplasm into a protrusion shape on the surface 11 of the porous thin film 10, and then the tip of the protrusion formed on the megakaryocyte M is torn off to form a culture solution as platelet P Released in S.

  The culture solution S used for culturing the megakaryocyte M is not particularly limited as long as it is a solution capable of producing platelets P from the megakaryocyte M in the culture solution S. The method for flowing the culture solution S through the microchannel 30 is not particularly limited. For example, the apparatus 1 includes a liquid feeding unit that generates a driving force for flowing the culture solution S through the microchannel 30. It is good as well. The liquid feeding unit may be a pump for pumping the culture solution S, for example.

  In addition, the present apparatus 1 is connected to, for example, an upstream flow path portion that is connected to the upstream end of the micro flow path 30 and allows the culture medium S to flow into the micro flow path 30, and a downstream end of the micro flow path 30. It is good also as providing the downstream flow path part for making the said culture solution S (for example, culture solution S containing platelets P) flow out from the said microchannel 30. FIG. The upstream channel portion and the downstream channel portion may be, for example, resin tubes.

  Further, the present apparatus 1 is connected to, for example, an upstream end of an upstream flow path section, and is connected to an upstream container section that contains a culture medium S that flows into the micro flow path 30 and a downstream end of the downstream flow path section. It is good also as providing the downstream container part which accommodates the said culture solution S (for example, culture solution S containing platelets P) which flows out out of the microchannel 30. FIG. The upstream container part and the downstream container part may be, for example, resin or glass containers.

  In this method, the platelets P released from the megakaryocytes M into the culture solution S in the microchannel 30 through the micropores 13 of the porous thin film 10 are collected. That is, the megakaryocyte M is disposed on the surface 11 of the porous thin film 10 where the micropores 13 are opened, and the micropores 13 are formed in a shape and size through which the platelets P can pass. Platelets P produced from the megakaryocytes M are released into the culture medium S flowing through the microchannel 30 via the micropores 13.

  Note that the release of platelets P through the micropores 13 causes platelets P produced in the culture chamber 20 or in the micropores 13 of the porous thin film 10 to pass through the micropores 13 and be released into the microchannel 30. And / or platelet P may be released from a part of the cytoplasm of the megakaryocyte M protruding into the microchannel 30 via the micropore 13.

  The platelets P released into the culture medium S flowing in the microchannel 30 are collected from the downstream end in the flow direction X of the microchannel 30. That is, the culture solution S containing platelets P produced from the megakaryocytes M is collected from the downstream end of the microchannel 30 to collect the platelets P.

  According to the apparatus 1 and the method described above, platelets P can be effectively mass-produced from megakaryocytes M outside the body. That is, by using the porous thin film 10 in which a plurality of micropores 13 are formed in a controlled shape and arrangement, the arrangement of the megakaryocytes M on the surface 11 on one side of the porous thin film 10 is directly controlled. In addition, by flowing the culture solution S in contact with the surface 12 on the other side of the porous thin film 10 in the microchannel 30, the shear stress is effectively applied to the appropriately arranged megakaryocytes M, and platelets are obtained. P can be produced efficiently.

  For example, a porous thin film 10 having a thickness of 5 to 40 μm in which a plurality of micropores 13 having an average pore diameter of 2 to 15 μm are formed with a hole diameter processing accuracy of ± 10% or less and an aperture ratio of 4 to 45% by microfabrication of the substrate, and the porous Using this apparatus 1 including a culture chamber 20 provided on one side of the thin film 10 and a microchannel 30 provided on the other side of the porous thin film 10, the porous thin film is formed in the culture chamber 20. When the megakaryocyte M is arranged on the surface 11 on the one side and the megakaryocyte M is cultured in the culture chamber 20 while flowing the culture medium S through the microchannel 30, the shear stress is applied to the megakaryocyte M. Is effectively produced, and the platelet P released into the culture medium S in the microchannel 30 through the micropores 13 can be efficiently recovered.

  In this case, the average pore diameter of the plurality of micropores 13 may be 2 to 10 μm, or 2 to 5 μm. Moreover, the aperture ratio of the plurality of micropores 13 may be 4 to 35%, or may be 4 to 25%. Moreover, the thickness of the porous thin film 10 may be 5 to 30 μm, or 5 to 20 μm.

  In addition, for example, megakaryocytes M derived from iPS cells or megakaryocyte M progenitor cells (for example, hematopoietic progenitor cells) are cryopreserved in advance, and the megakaryocytes M or progenitor cells are thawed at an arbitrary timing and left as they are. By culturing in the apparatus 1, a large amount of platelets P can be produced in vitro in a short period of time. Therefore, according to this apparatus 1 and this method, it can also be expected to industrially produce a large amount of platelets P from megakaryocytes M in vitro.

  In addition, the device 1 may further include a temperature control unit that controls the temperature in the vicinity of the porous thin film 10. This temperature control part is good also as controlling the temperature of the porous thin film 10 vicinity to 30-40 degreeC, for example.

  The temperature control unit is not particularly limited as long as it controls the temperature in the vicinity of the porous thin film 10. That is, the temperature control unit that controls the temperature in the vicinity of the porous thin film is, for example, a temperature control unit that controls the temperature of the culture solution S in the culture chamber 20 and / or the culture solution S in the microchannel 30 of the apparatus 1. It may be there.

  Specifically, the temperature control unit is, for example, a temperature sensor (for example, a thermocouple) that measures the temperature of the culture solution S stored in the culture chamber 20 of the apparatus 1 and / or the culture solution S flowing through the microchannel 30. And a heater for heating the culture medium S based on the temperature measurement result by the temperature sensor. In this case, the temperature control unit sets the temperature of the culture solution S in the culture chamber 20 and / or the culture solution S in the microchannel 30 of the apparatus 1 to a predetermined value suitable for the production of platelets P from the megakaryocytes M. It can be maintained within the temperature range. This temperature range is good also as being 30-40 degreeC, for example.

  In addition, the temperature sensor may be provided so that, for example, the detection unit (for example, the tip of the temperature sensor) is immersed in the culture solution S in the culture chamber 20. In this case, for example, during the culture of the megakaryocyte M, the temperature of the culture solution S in which the megakaryocyte M is immersed is rapidly increased by immersing the detection unit in the culture solution S in the culture chamber 20. And it can measure accurately. By providing the apparatus 1 with the temperature control unit as described above, the megakaryocyte M can be reliably and stably cultured at a temperature suitable for the production of platelets P.

  Next, specific examples according to the present embodiment will be described.

[Manufacture of platelet production channel device]
In this example, this device 1 as shown in FIGS. 2A and 2B was manufactured as a platelet production channel device using a MEMS processing technique. FIG. 2A shows a cross section of the device 1 and FIG. 2B shows the device 1 in plan view. Note that FIG. 2B shows a state in which the lid 23 that closes the opening above the culture chamber 20 of the apparatus 1 is removed.

  In this embodiment, as shown in FIG. 3, a plurality of micro holes 13 are formed in a part of the substrate by micro-processing of the substrate, which will be described later, and the part is cut to reduce the thickness. The substrate part 40 integrally having the porous thin film 10 and the frame part 14 which is another part of the substrate surrounding the porous thin film 10 was manufactured. As the substrate 100, a Si wafer was used, and a porous thin film 10 having a thickness of 10 μm was formed in a rectangular area (2 mm × 2 mm) in the center of the substrate 100.

  In the porous thin film 10, a plurality of micropores 13 having an average pore diameter of 4 μm are arranged with a constant hole distance accuracy of ± 10% or less (the center of one micropore 13 of two adjacent micropores 13. And the distance between the center of the other micropore and the other micropores).

  The regular arrangement of the plurality of micro holes 13 was a honeycomb arrangement as shown in FIG. 4A. For example, as shown in FIG. 4B, the arrangement of the plurality of micro holes 13 is a lithography / etching process such as a shift type arrangement in which the micro holes 13 nearest to the crystal axis direction of Si constituting the substrate are not arranged. It is not particularly limited as long as it is an intentional arrangement controlled by technology.

  5A to 5L show steps included in the formation of the porous thin film 10 by fine processing of the substrate 100 made of an inorganic material. As the substrate 100, a general SOI (Silicon on Insulator) wafer, which is a kind of silicon wafer, was used.

  As shown in FIG. 5A, the substrate 100 had a BOX (Buried Oxide) layer 102 and an SOI layer 103 stacked on a Si substrate 101. Hereinafter, FIGS. 5B to 5K will be described by focusing on the portion of the substrate 100 surrounded by the alternate long and short dash line V shown in FIG. 5A.

  First, as shown in FIG. 5B, the resist 110 applied to the surface 100a of the substrate 100 (the surface of the SOI layer 103) was formed by exposing the arrangement pattern (arrangement shown in FIG. 4A) of the plurality of micro holes 13 to each other.

Next, as shown in FIG. 5C, the SOI layer 103 was dry-etched according to the formed arrangement pattern. The dry etching was performed by inductively coupled plasma reactive ion etching (ICP-RIE) using a general Bosch process using SF ₆ and C ₄ F ₈ .

After the etching, as shown in FIG. 5D, the resist 110 was removed using a resist stripping solution or the like. After removing the resist, as shown in FIG. 5E, the entire surface of the substrate 100 is covered as a hard mask 120 by a plasma CVD method (a general RF plasma CVD method using SiH ₄ and N ₂ O gas at about 300 ° C.). A SiO ₂ film having a thickness of about 3 μm was formed. Further, as shown in FIG. 5F, a resist 111 was applied as a protective film on the hard mask 120 on the SOI layer 103 side.

  Then, as shown in FIG. 5G, the substrate 100 was turned upside down, and a resist 112 was applied on the hard mask 120 on the back surface 100b (see FIG. 5A) side of the substrate 100. Thereafter, the resist 112 is exposed so that a rectangular porous thin film 10 (2 mm × 2 mm) is finally formed, and a rectangular groove having a size corresponding to the rectangle and allowing for the thickness of the Si substrate 101 is formed. Formed.

Next, as shown in FIG. 5H, the SiO ₂ film as the hard mask 120 was etched using HF water (hydrogen fluoride water) or the like according to the shape of the groove. After the etching, as shown in FIG. 5I, the resist 112 was removed using a resist stripping solution or the like.

  After the resist removal, as shown in FIG. 5J, the Si substrate 101 was wet-etched so as to stop at the BOX layer 102 in accordance with the shape of the groove. The wet etching was performed by a general silicon wet etching process using TMAH (tetramethylammonium hydroxide) or KOH (potassium hydroxide).

Further, as shown in FIG. 5K, the BOX layer 102 and the hard mask 120 (SiO ₂ film) were removed by wet etching using HF water or the like in a rectangular area where the porous thin film 10 was formed. As a result, as shown in FIG. 5K, a porous thin film 10 having a plurality of micropores 13 formed in a regular arrangement was obtained.

  Finally, as shown in FIG. 5L, a substrate portion 40 (see FIG. 3), which is a Si chip integrally including the porous thin film 10 and the frame portion 14, was cut out from the substrate 100 by dicing. In FIG. 5L, the portion surrounded by the alternate long and short dash line V corresponds to one substrate portion 40.

  The microchannel 30 was formed by laminating a plurality of quartz chips formed with holes and grooves. The microchannel 30 was formed in a linear shape having a length in the flow direction X of more than 2 mm, a width (length in the width direction) of 2 mm, and a height of 0.3 mm. That is, as shown in FIG. 2A and FIG. 2B, the microchannel 30 was formed so that the culture solution S flowed linearly while contacting the entire surface 12 of the porous thin film 10. In addition, it is good also as replacing with quartz for forming the microchannel 30 using other materials, such as borosilicate glass.

  Further, as shown in FIG. 2A, the culture chamber 20 is formed by laminating a metal upper holder 21 in which a through hole is formed at a position corresponding to the porous thin film 10 on the substrate portion 40 having the porous thin film 10. Formed. When the megakaryocyte M was cultured in the culture chamber 20, the opening above the through hole of the upper holder 21 was closed with a lid 23 as shown in FIG. 2A.

  Further, on the lower side of the microchannel 30, a metal lower holder 22 in which through holes are formed at positions corresponding to the respective openings at the upstream end and the downstream end of the microchannel 30 was laminated. Then, as shown in FIGS. 2A and 2B, the upper holder 21 and the lower holder 22 sandwiching the substrate portion 40 having the porous thin film 10 and the microchannel 30 were fixed with a fixture 50 (bolts and nuts). A sealing material (O-ring) is disposed between the stacked components (for example, between the substrate section 40 and the microchannel 30 and the upper holder 21 and between the microchannel 30 and the lower holder 22). This prevented liquid leakage.

  In addition, a silicon tube (not shown) was connected to the upstream end of the microchannel 30 via the lower holder 22 as an upstream channel section. Furthermore, a glass bottle (not shown) in which a fresh culture solution S that flows into the microchannel 30 was accommodated was connected to the upstream end of the upstream channel section as an upstream container section.

  On the other hand, a silicon tube (not shown) was connected to the downstream end of the microchannel 30 via the lower holder 22 as a downstream channel section. Furthermore, a glass bottle (not shown) for accommodating the culture medium S collected from the microchannel 30 was connected to the downstream end of the downstream channel section as a downstream container section. Moreover, the syringe pump for pumping the culture solution S was connected to the silicon tube as a liquid feeding part.

[Production of platelets]
A culture solution S containing megakaryocytes M derived from iPS cells is injected into the culture chamber 20, and the megakaryocytes M are precipitated in the culture solution S, thereby arranging the megakaryocytes M on the surface 11 of the porous thin film 10. did. As the culture solution S, a Hepes-Tyrode solution to which 0.1% of BSA (Bovine Serum Albumin) was added was used.

  Next, the liquid feeding unit is operated to cause the culture solution S to flow into the microchannel 30 via the upstream channel unit, and to cause the culture solution S to flow out of the microchannel 30 via the downstream channel unit. As a result, the culture of the megakaryocytes M was started in the culture chamber 20 while flowing the culture medium S through the microchannel 30.

  The flow rate of the culture solution S was 380 μL / min. As a result, the flow rate of the culture solution S in the microchannel 30 was about 10 mm / second. Then, the culture solution S was allowed to flow through the microchannel 30 for 8 hours, and the culture solution S flowing out from the microchannel 30 was collected in the downstream container portion. As a comparative example, 50 mL of the culture solution S containing megakaryocytes M derived from iPS cells was placed in a petri dish, and the megakaryocytes M were statically cultured for 8 hours.

  FIG. 6 shows an example of the result of placing megakaryocytes M prestained with a fluorescent dye on the surface 11 on one side of the porous thin film 10 and observing with a fluorescence microscope from the other side of the porous thin film 10. As shown in FIG. 6, megakaryocytes M that were fluorescently stained and arranged on the surface 11 of the porous thin film 10 were observed through the micropores 13.

  FIG. 7 shows the culture conditions (cultured cells, culture cells, and examples) in which platelet P was produced from megakaryocytes M by the present method using this device 1 and a comparative example in which platelet P was produced from megakaryocytes M in a petri dish. The culture solution, the amount of the collected culture solution, the culture time and the culture temperature) and the results of evaluating the production amount of platelets P are shown.

  The amount of platelet P produced was calculated by calculating the ratio of the number of collected platelets P to the number of cultured megakaryocytes M for each of the examples and comparative examples, and further calculating the ratio of the comparative example as “1”. In this case, the ratio was evaluated by calculating the ratio.

  As a result, as shown in FIG. 7, in the example, 86 times as many platelets P as the comparative example could be produced. That is, according to this method using this apparatus 1, it was confirmed that the production of platelets P from megakaryocytes M in vitro was significantly promoted and the platelets P could be produced in large quantities.

  FIG. 8 shows another example of the apparatus 1. In the example shown in FIG. 8, the apparatus 1 includes a temperature control unit that controls the temperature of the culture solution S in the culture chamber 20 and / or the culture solution S in the microchannel 30. A temperature sensor 60 (for example, a thermocouple) that measures the temperature of the culture solution S in the culture chamber 20 and a heater that heats the culture solution S based on the temperature measurement result by the temperature sensor 60 are provided.

  Since the temperature sensor 60 is provided so that the detection part at the tip thereof protrudes into the culture chamber 20 and is immersed in the culture solution S containing the megakaryocytes M in the culture chamber 20, the temperature of the culture solution S Can be measured accurately and quickly.

  Then, the temperature control unit heats the culture solution S flowing into the micro flow channel 30 with a heater (not shown) based on the temperature measurement result by the temperature sensor 60, as necessary. Is maintained in a predetermined temperature range (for example, 30 to 40 ° C.) suitable for production of platelets P from megakaryocytes M.

  DESCRIPTION OF SYMBOLS 1 Platelet production flow path apparatus, 10 Porous thin film, 11 One side surface, 12 The other side surface, 13 Micropore, 14 Frame part, 20 Culture chamber, 21 Upper holder, 22 Lower holder, 23 Lid part, 30 Microflow Path, 40 substrate part, 50 fixture, 60 temperature sensor, 100 substrate, 100a surface, 100b back surface, 101 Si substrate, 102 BOX layer, 103 SOI layer, 110, 111, 112 resist, 120 hard mask, M megakaryocyte, P platelets, S medium.

Claims (11)

A porous thin film in which a plurality of micropores are formed in a controlled shape and arrangement, and megakaryocytes are arranged on the surface on one side thereof;
A culture chamber provided on the one side of the porous thin film in which the megakaryocytes are cultured;
A microchannel provided on the other side of the porous thin film, through which a culture solution flows while the megakaryocytes are cultured in the culture chamber;
A platelet production channel device comprising: The platelet production channel device according to claim 1, wherein each of the plurality of micropores has a pore diameter of 2 to 15 µm. The platelet production channel device according to claim 1 or 2, wherein an average pore diameter of the plurality of micropores is 2 to 15 µm. The platelet production channel device according to any one of claims 1 to 3, wherein the plurality of micropores are formed with a hole diameter processing accuracy of ± 10% or less. The platelet production channel device according to any one of claims 1 to 4, wherein the porous thin film has a thickness of 5 to 40 µm. The platelet production channel device according to any one of claims 1 to 5, wherein an opening ratio of the plurality of micropores is 4 to 45%. The platelet production channel device according to any one of claims 1 to 6, wherein the microchannel is formed so that the culture solution can flow at a flow rate of 1 to 100 mm / sec. The platelet production channel device according to any one of claims 1 to 7, further comprising a temperature control unit that controls a temperature in the vicinity of the porous thin film. The platelet production channel device according to claim 8, wherein the temperature control unit controls the temperature to 30 to 40 ° C. A porous thin film having a plurality of micropores formed in a controlled shape and arrangement, a culture chamber provided on one side of the porous thin film, and a microchannel provided on the other side of the porous thin film Preparing a platelet production channel device,
Arranging a megakaryocyte on the surface of the one side of the porous thin film in the culture chamber;
Culturing the megakaryocyte in the culture chamber while flowing a culture solution in the microchannel to produce platelets; and the platelets released into the culture solution in the microchannel through the micropores Collecting,
A method for producing platelets, comprising: The method for producing platelets according to claim 10, wherein the culture solution is caused to flow through the microchannel at a flow rate of 1 to 100 mm / sec.

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