WO2020003504A1 - Système de culture par perfusion - Google Patents

Système de culture par perfusion Download PDF

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Publication number
WO2020003504A1
WO2020003504A1 PCT/JP2018/024870 JP2018024870W WO2020003504A1 WO 2020003504 A1 WO2020003504 A1 WO 2020003504A1 JP 2018024870 W JP2018024870 W JP 2018024870W WO 2020003504 A1 WO2020003504 A1 WO 2020003504A1
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Prior art keywords
culture
vessel unit
flow path
container
culture solution
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PCT/JP2018/024870
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English (en)
Japanese (ja)
Inventor
学司 加藤
順一 桑原
直史 小出
Original Assignee
株式会社サンプラテック
株式会社ビジョンケア
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Application filed by 株式会社サンプラテック, 株式会社ビジョンケア filed Critical 株式会社サンプラテック
Priority to JP2020526860A priority Critical patent/JP7162226B2/ja
Priority to PCT/JP2018/024870 priority patent/WO2020003504A1/fr
Publication of WO2020003504A1 publication Critical patent/WO2020003504A1/fr

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    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a system for culturing cells and biological tissues, and more particularly to a perfusion culture system capable of supplying a new culture solution to a closed culture vessel.
  • a perfusion culture apparatus which eliminates the labor of replacing the culture medium (medium) by human hands, automatically supplies the culture medium at a constant speed, and simultaneously withdraws the same amount of the culture medium (for example, see Patent Reference 2).
  • these perfusion culture systems basically include a pump drive unit with a power supply and a control controller for exchanging a culture solution and controlling the culture solution, so that the equipment is large.
  • a pump drive unit with a power supply
  • a control controller for exchanging a culture solution and controlling the culture solution, so that the equipment is large.
  • there is a problem of a risk of failure of an electric system by bringing the device into an incubator in a severe and humid environment.
  • the present invention has been conceived under such circumstances, and provides a perfusion culture system that is compact and is suitable for eliminating the risk of failure, realizing labor saving of workers and ensuring cell quality. Is the main task.
  • the present invention employs the following technical means.
  • the perfusion culture system provided by the present invention is a closed culture container unit having a fluid inlet and a fluid outlet, a culture solution container capable of storing a culture solution, and a vacuum container whose inside is adjusted to a negative pressure.
  • a first flow path one end of which can communicate with the inside of the culture vessel unit via the fluid inlet, and the other end of which can communicate with the inside of the culture solution container;
  • a second flow path communicable with the inside of the culture vessel via an outlet, and the other end of which is communicable with the inside of the vacuum vessel unit.
  • a constriction is provided in at least one of the first flow path and the second flow path.
  • the constricted portion is a columnar body through which fine holes penetrate.
  • the constriction is a microchannel chip.
  • the culture vessel unit and the culture solution container are provided in the first flow path, and the culture vessel unit and the culture solution container are connected in a sealed state, and the culture container unit and the culture solution container are sealed with each other.
  • a detachable first connection means provided in the second flow path, connects and connects the culture vessel unit and the vacuum vessel unit in a sealed state, and keeps the culture vessel unit and the vacuum vessel unit in a sealed state with each other.
  • second connection means that can be separated.
  • a plurality of the culture solution containers are provided, and the first flow path has a first main path communicating with the culture container unit and a plurality of first main channels each communicating with the culture solution container. And an end of the first main road and an end of each of the plurality of first branch roads are connected, and one of the plurality of first branch roads and the first main road are connected to each other.
  • a first switching unit capable of switching a flow path so as to communicate with a road is provided.
  • a plurality of the vacuum vessel units are provided, and the second flow path is provided with a second main path communicating with the culture vessel unit, and a plurality of second branches each communicating with the vacuum vessel unit. And an end of the second main road and an end of each of the plurality of second branch roads are connected, and one of the plurality of second branch roads and the second main road are connected to each other. And second switching means capable of switching the flow path so that the communication can be established.
  • the vacuum vessel unit includes a bottomed tubular body having an open end, and a stopper for closing the open end, and is attached to the other end of the second flow path, A puncture needle capable of piercing the stopper is provided.
  • the culture vessel unit has a bottomed cylindrical vessel body having an opening at one end in a first direction, and is mounted on the vessel body and covers the inside of the vessel body by closing the opening.
  • An attachment that seals a space, wherein the attachment is configured such that the one end communicates with the fluid inflow port that communicates with the outside, and the other end communicates with the fluid-introducing channel that communicates with the sealed inside space, and the fluid includes one end that communicates with the outside.
  • a fluid outlet channel that communicates with the inner space, the other end of which is closed.
  • the attachment has a protrusion that fits inside the container body and protrudes toward the other side in the first direction.
  • the culture solution container is sealed, has flexibility, and can change its volume.
  • the second flow path allows a flow of a fluid from the culture vessel unit side to the vacuum vessel unit side, and a fluid flows from the vacuum vessel unit side to the culture vessel unit side.
  • a check valve for preventing the flow of air is provided.
  • the inner surface of the culture vessel unit includes a flat cell culture surface, and the cell culture surface is subjected to a surface treatment for improving cell adhesion.
  • FIG. 2 is an exploded view of the perfusion culture system shown in FIG. 1. It is a top view which shows an example of a culture container unit.
  • FIG. 4 is a sectional view taken along the line IV-IV in FIG. 3.
  • FIG. 5 is a sectional view taken along line VV in FIG. 3.
  • FIG. 4 is an exploded perspective view of the culture vessel unit shown in FIG.
  • FIG. 6 is a cross-sectional view similar to FIG. 5, showing a state in which contents are accommodated in a culture vessel unit.
  • FIGS. 1 to 6 show a first embodiment of a perfusion culture system according to the present invention.
  • the perfusion culture system A10 of the present embodiment includes a culture vessel unit 1, a culture solution container 2, a vacuum vessel unit 3, a first flow path 41, a second flow path 42, a first connection means 51, It has a second connection means 52 and a constricted part 6.
  • the perfusion culture system A10 supplies the new culture solution contained in the culture solution container 2 to the culture container unit 1 and collects the old culture solution in the culture container unit 1 into the vacuum container unit 3. It is for doing.
  • the culture vessel unit 1 includes a vessel body 11 and an attachment 12.
  • a vessel body 11 As the container body 11, an existing open culture container such as a dish (dish) or a well plate is used, and the present embodiment shows a case where the container body 11 is a dish.
  • the container body 11 has a bottomed cylindrical shape having an opening 110 at an upper end (one end in the first direction), and has a side plate 111 and a bottom plate 112.
  • the upper surface of the bottom plate 112 is a flat cell culture surface for culturing cells.
  • the cell culture surface (the upper surface of the bottom plate 112) is appropriately subjected to a surface treatment for improving cell adhesion as needed.
  • Examples of the surface treatment include a corona discharge treatment and a plasma treatment (hydrophilization treatment).
  • the attachment 12 is attached to the container main body 11 and is for closing the opening 110 of the container main body 11 to seal the inner space of the container main body 11.
  • the attachment 12 includes a base member 12A and a seal member 12B.
  • the base member 12 ⁇ / b> A has a bottom wall 121, a cylindrical side wall 122 extending upright from the periphery of the bottom wall 121, and a flange 123.
  • the bottom wall 121 fits inside the side plate 111 of the container main body 11 with an interval from the side plate 111 and protrudes downward (the other side in the first direction).
  • the bottom wall 121 corresponds to a protrusion in the present invention.
  • the flange 123 extends radially outward from the upper end of the side wall 122 and has an annular shape.
  • the base member 12A having such a configuration overlaps with all of the openings 110 of the container body 11 in a plan view.
  • the container body 11 and the base member 12A are formed of, for example, a translucent or transparent hard plastic material.
  • a material having transparency such as polycarbonate, cycloolefin polymer, and cycloolefin copolymer, is preferably used, in addition to polystyrene and methylpentene, but is not limited thereto.
  • the bottom plate 112 of the container body 11 and the bottom wall 121 of the base member 12A are transparent.
  • the bottom plate 112 and the bottom wall 121 are portions where the inner space of the container body 11 can be visually recognized from the outside.
  • the seal member 12B is, for example, a rubber molded product, and includes a vertically extending cylindrical portion 125, a flange portion 126, and an annular protrusion 127, as shown in FIGS.
  • the seal member 12B is interposed between the side plate 111 of the container body 11 and the side wall 122 or the flange 123 of the base member 12A.
  • the cylindrical portion 125 is fitted on the side wall 122 of the base member 12A.
  • the inner diameter of the cylindrical portion 125 in the natural state is slightly smaller than the outer diameter of the side wall 122, so that the cylindrical portion 125 (the seal member 12B) is pressed against the side wall 122 (the base member 12A).
  • the flange portion 126 extends radially outward from the upper end of the cylindrical portion 125 and has an annular shape.
  • the flange portion 126 overlaps the flange 123 of the base member 12A in a plan view.
  • the annular projection 127 protrudes radially outward from the outer periphery of the cylindrical portion 125 and is in pressure contact with the inner peripheral surface of the side plate 111 when the attachment 12 is mounted on the container body 11.
  • the seal member 12B is made of a material having flexibility and elasticity.
  • the material forming the seal member 12B include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Although details will be described later, in consideration of the contact between the sealing member 12B and the contents (for example, culture solution), a medical silicone rubber having no cytotoxicity and biocompatibility is more preferable as the material of the sealing member 12B. .
  • the hardness of the seal member 12B is preferably, for example, about 20 to 40 degrees with a rubber hardness.
  • the attachment 12 has a fluid introduction channel 13 and a fluid outlet channel 14.
  • the fluid introduction flow path 13 is a flow path for introducing a fluid such as a culture solution from the outside to the inside of the culture vessel unit 1, and in the present embodiment, is constituted by holes and grooves formed in the seal member 12 ⁇ / b> B. More specifically, one end of the fluid introduction flow channel 13 is a fluid inlet 131 that communicates with the outside, and the other end communicates with the inner space of the sealed container body 11.
  • the fluid outlet channel 14 is a channel for leading the fluid from the inside of the culture vessel unit 1 to the outside.
  • the fluid outlet channel 14 is provided on the opposite side of the fluid inlet channel 13 across the center of the attachment 12 in plan view.
  • the fluid outlet channel 14 is constituted by a hole or a groove formed in the seal member 12B, and has one end serving as a fluid outlet 141 communicating with the outside, and the other end communicating with the inner space of the sealed container body 11. ing.
  • the culture solution container 2 shown in FIG. 1 is a sealed container for storing a replacement culture solution.
  • the culture solution container 2 is provided with an outlet 21 for extracting the contents.
  • the culture solution container 2 is preferably configured to be flexible and changeable in volume. Examples of such a culture solution container 2 include a bellows-like container, a film-like container, a syringe, a laminated peeling bottle, and the like. In this embodiment, the case where the culture solution container 2 is a bellows-like container is shown.
  • the vacuum container unit 3 is adjusted to a negative pressure state, and collects a culture solution and the like existing in the culture container unit 1.
  • the vacuum container unit 3 is configured to include a tube 31 and a stopper 32.
  • the tube 31 has a bottomed cylindrical shape having an open end 311 at the tip.
  • the plug 32 closes the open end 311, and the inside of the sealed tube 31 is adjusted to a negative pressure state.
  • Part of the stopper 32 is made of, for example, a rubber material capable of piercing a puncture needle.
  • a general-purpose blood collection tube can be used as the vacuum container unit 3 having such a configuration.
  • the first flow path 41 is a fluid flow path that connects between the culture vessel unit 1 and the culture solution container 2, and is formed of, for example, a flexible tube. One end of the first flow path 41 can communicate with the inside of the culture vessel unit 1 via the fluid inlet 131. The other end of the first flow path 41 can communicate with the inside of the culture solution container 2 via the outlet 21.
  • the first connection means 51 shown in FIG. 1 is provided in the first flow path 41, and connects and connects the culture vessel unit 1 and the culture solution container 2 in a sealed state. Further, the first connection means 51 can separate the culture vessel unit 1 and the culture solution container 2 from each other in a sealed state (see FIG. 2).
  • the first connection means 51 having such a configuration is realized by a luer lock connection structure having a female luer connector 511 and a male luer connector 512, for example.
  • the first flow path 41 has a partial flow path 411 connected to the culture vessel unit 1 (fluid inlet 131) and a partial flow path connected to the culture solution container 2 (outlet 21). Road 412.
  • a female luer connector 511 is provided at the tip of the partial flow path 411
  • a male luer connector 512 is provided at the tip of the partial flow path 412.
  • the second flow path 42 is a fluid flow path connecting the culture vessel unit 1 and the vacuum vessel unit 3, and is made of, for example, a flexible tube.
  • One end of the second flow path 42 can communicate with the inside of the culture vessel unit 1 via the fluid outlet 141.
  • a puncture needle 43 that can puncture the stopper 32 of the vacuum container unit 3 is attached to the other end of the second channel 42.
  • the other end of the second flow path 42 can communicate with the inside of the vacuum container unit 3 (tube 31) via the puncture needle 43.
  • the second connection means 52 shown in FIG. 1 is provided in the second flow path 42 and connects and connects the culture vessel unit 1 and the vacuum vessel unit 3 in a sealed state. Further, the second connection means 52 can separate the culture vessel unit 1 and the vacuum vessel unit 3 from each other in a sealed state (see FIG. 2).
  • the second connection means 52 having such a configuration is realized by a luer lock connection structure having a female luer connector 521 and a male luer connector 522, for example.
  • the second flow path 42 includes a partial flow path 421 connected to the culture vessel unit 1 (the fluid outlet 141) and a partial flow path 422 connected to the vacuum vessel unit 3. You.
  • a female luer connector 521 is provided at the tip of the partial flow path 421, and a male luer connector 522 is provided at the tip of the partial flow path 422.
  • the puncture needle 43 is attached to the end of the partial flow path 422.
  • the puncture needle 43 is surrounded by a cylindrical holder 44 to prevent danger.
  • the inner diameter of the holder 44 is larger than the outer diameter of the tube 31, and, as understood from FIGS. 1 and 2, by inserting the tube 31 (vacuum container unit 3) into the holder 44, Puncture needle 43 can be safely punctured.
  • the constriction 6 is provided in at least one of the first flow path 41 and the second flow path 42.
  • the constricted portion 6 has a portion where the cross section of the flow path is narrowed, and the flow rate of the fluid passing through the constricted portion 6 per unit time (hereinafter, simply referred to as “flow rate”) is adjusted to be desired. Is what you do.
  • the constricted portion 6 is formed of a columnar body having fine holes penetrating in the longitudinal direction.
  • the diameter of the micropores for example, about 0.02 to 0.05 mm
  • the length of the micropores the length in the longitudinal direction: for example, about 5 to 20 mm
  • the flow rate of a culture solution or the like can be adjusted.
  • the constricted portion 6 having such a configuration can be manufactured by extrusion molding and sintering using a ceramic material such as zirconia, for example, and it is possible to obtain a high-accuracy fine through hole with a high aspect ratio. As shown in FIGS.
  • the constricted portion 6 is provided in a partial flow path 422 of the second flow path 42.
  • the constricted portion 6 may be provided with a tapered portion or a large-diameter portion at its end so as to facilitate connection with the partial flow path 422 (flexible tube) and to make it difficult to separate.
  • the perfusion culture system A10 accommodates cultured cells and a culture solution inside a closed culture container unit 1 configured using an open culture container (vessel main body 11), and maintains the cell culture state while maintaining the cell culture state. Used to make exchanges.
  • the culture cells and culture solution contained in the container body 11 (culture container unit 1) are not particularly limited.
  • the culture container unit 1 In the cell culture using the perfusion culture system A10, first, cells to be cultured are seeded in the container main body 11 (culture container unit 1), and the culture container unit 1 is filled with a culture solution.
  • the seeding of the cells may be performed by removing the attachment 12 from the container main body 11 and directly injecting a culture solution containing cells into the container main body 11, but may also be performed using the perfusion culture system A10. .
  • FIG. 7 shows a state where the inside of the container body 11 (culture container unit 1) is filled with the culture solution M1. After the culture vessel unit 1 is filled with the culture solution, the connection between the culture vessel unit 1 and the culture solution container 2 and the connection between the culture vessel unit 1 and the syringe are separated.
  • the cells in the culture vessel unit 1 adhere to the cell culture surface (the upper surface of the bottom plate 112) over time, and the proliferation starts. If the upper surface of the bottom plate 112 has been subjected to the above-described surface treatment for improving cell adhesion, the cells will adhere to the cell culture surface more reliably. Therefore, such a configuration is preferable for appropriately progressing cell culture in the culture vessel unit 1.
  • the bottom plate 112 of the container body 11 and the bottom wall 121 of the base member 12A are transparent. Thereby, the state inside the culture vessel unit 1 can be observed from either the upper part or the lower part of the culture vessel unit 1 via the transparent bottom wall 121 or the bottom plate 112. With such a configuration, irradiation light can be transmitted regardless of whether the light source is above or below, and cells can be observed using various types of microscopes such as an optical microscope and a phase contrast microscope.
  • the bottom wall 121 of the base member 12A protrudes downward.
  • the culture solution in the culture vessel unit 1 comes into contact with the surface of the bottom wall 121.
  • the liquid surface of the culture solution becomes flat due to the underwater spectacle effect, and irregular reflection of irradiation light can be suppressed, and an effect such as easy focusing of the microscope can be expected.
  • the culture vessel unit 1 and the culture solution container 2 communicate with each other via the first flow path 41, and the culture vessel unit 1 and the vacuum vessel unit 3 communicate with each other via the second flow path 42.
  • the old culture solution in the culture vessel unit 1 moves into the vacuum vessel unit 3 via the second flow path 42 by the suction force of the vacuum vessel unit 3 (tube 31) in which the inside is in a negative pressure state
  • the new culture solution in the culture solution container 2 moves into the culture vessel unit 1 via the first flow path 41.
  • the first connection means 51 and the second connection means 52 are returned from the connected state to the separated state.
  • the culture solution in the culture vessel unit 1 can be exchanged without using an electric device such as a controller or a power supply. Therefore, the perfusion culture system A10 can be downsized without any fear of failure.
  • the culture solution in the culture vessel unit 1 can be exchanged by a one-touch operation such as changing the first connection means 51 and the second connection means 52 from the separated state to the connected state as described above.
  • the culture medium can be exchanged in a short time by selecting the pore diameter and the length of the stenosis portion 6, or the culture medium can be supplied to the culture system (culture vessel unit 1) slowly at a constant supply rate over several days.
  • the same amount of culture solution can be withdrawn from the culture vessel unit 1 (so-called perfusion culture).
  • the culture solution exchange operation can be performed while the culture container unit 1 is in the closed state, and the risk of contamination can be avoided. Therefore, according to the perfusion culture system A10, it is possible to reduce the operation burden on the operator when exchanging the culture solution, and to realize the quality of the cells.
  • the suction amount of the culture solution into the tube 31 is determined in advance. It is possible to keep.
  • the stenosis portion 6 is provided in the second flow path 42 connecting the culture vessel unit 1 and the vacuum vessel unit 3. According to the configuration including the stenosis part 6, the flow rate of the culture solution exchange can be adjusted. With such a stenotic portion 6, the exchange time of the culture solution can be appropriately set within a range of, for example, about 1 to 48 hours according to the exchange amount of the culture solution. Although the flow rate suitable for the exchange of the culture solution varies depending on the type of the cultured cells, the exchange amount of the culture solution, and the like, according to the present embodiment, the flow rate at the time of the exchange of the culture solution can be appropriately adjusted.
  • the luer lock connection portion may not be provided.
  • the second flow path 42 one end is connected to the culture vessel unit 1 (fluid outlet 141), and the other end is provided with a puncture needle 43 attached thereto.
  • the exchange of the culture solution may be started by puncturing the medium.
  • a check valve may be provided in the second flow path 42. The check valve allows the flow of fluid from the culture vessel unit 1 side to the vacuum vessel unit 3 side, and prevents the flow of fluid from the vacuum vessel unit 3 side to the culture vessel unit 1 side. According to such a configuration, it is possible to prevent a disadvantage that the old culture solution moved from the culture vessel unit 1 to the vacuum vessel unit 3 returns to the culture vessel unit 1 side.
  • the culture solution container 2 has flexibility and can change its volume. According to such a configuration, as the culture solution in the culture solution container 2 moves to the culture vessel unit 1, the internal volume of the culture solution container 2 decreases, and the culture solution exchange is performed smoothly.
  • the attachment 12 has a bottom wall 121 (projection), and the bottom wall 121 projects downward (the other side in the first direction) inside the container body 11. According to such a configuration, the accommodation space for the culture solution in the container body 11 is reduced, and the amount of the culture solution used in cell culture can be reduced.
  • the old culture medium in the culture vessel unit 1 is collected in the vacuum vessel unit 3 (tube 31). According to such a configuration, the collected culture solution can be easily sampled.
  • the cultured cells in the culture vessel unit 1 are collected.
  • the collection of the cultured cells may be performed by removing the attachment 12 from the container main body 11 and directly peeling the cultured cells in the container main body 11, but may also be performed using the perfusion culture system A10.
  • a culture solution container 2 containing a buffer solution is prepared, and the culture solution container 2 is connected to the culture container unit 1. connect. Further, a syringe is prepared in place of the vacuum container unit 3, and the syringe is connected to the culture container unit 1. Then, the buffer in the culture solution container 2 is guided into the culture container unit 1 by pulling the piston to make the inside of the syringe negative pressure, and the old culture solution in the culture container unit 1 is washed away.
  • a culture solution container 2 containing an enzyme (for example, trypsin) for peeling the cultured cells from the container body 11 is prepared, and the culture solution container 2 is connected to the culture container unit 1. Further, the vacuum container unit 3 is prepared, and the vacuum container unit 3 is connected to the culture container unit 1. Then, the enzyme in the culture solution container 2 moves into the culture vessel unit 1, and the cultured cells in the culture vessel unit 1 are peeled off by the enzyme treatment. The peeled cultured cells are collected in the vacuum container unit 3 via the second flow path 42.
  • a mesh-shaped filter may be interposed in the partial flow path 422 on the vacuum container unit 3 side instead of the constriction 6. . The filter removes unnecessary substances such as foreign substances and aggregated cells and functions as a strainer.
  • FIG. 8 shows a second embodiment of the perfusion culture system according to the present invention.
  • the perfusion culture system A20 of the present embodiment includes a culture vessel unit 1, a plurality (three in this embodiment) of culture solution containers 2, a plurality (three in this embodiment) of vacuum vessel units 3,
  • the apparatus includes a first flow path 41, a second flow path 42, a plurality of constrictions 6, and switching valves 7 and 8.
  • the same or similar elements as those of the above-described embodiment are denoted by the same reference numerals, and description thereof will be omitted as appropriate.
  • the perfusion culture system A20 of the present embodiment is significantly different from the above-described perfusion culture system A10 in that the perfusion culture system A20 includes a plurality of culture solution containers 2 and a plurality of vacuum container units 3. Accordingly, the connection structure between the culture solution container 2 or the vacuum container unit 3 and the culture container unit 1 is different from that of the above-described embodiment.
  • the configurations of the culture vessel unit 1, the culture solution container 2, and the vacuum vessel unit 3 are substantially the same as those in the above embodiment.
  • the first flow path 41 includes a first main path 415 and a plurality (three in this embodiment) of first branch paths 416.
  • One end of the first main route 415 is connected to the culture vessel unit 1 (the fluid inlet 131), and the other end of the first main route 415 is connected to the switching valve 7.
  • the plurality of first branch paths 416 correspond to each of the plurality of culture solution containers 2, each having one end communicating with the culture solution container 2 (the outlet 21), and the other end having the switching valve 7. It is connected to the.
  • the switching valve 7 selectively connects one of the plurality of culture solution containers 2 to the culture vessel unit 1. Although a detailed illustration is omitted, the switching valve 7 has a valve structure capable of switching a flow path such that any one of the plurality of first branch paths 416 and the first main path 415 communicate with each other. An operation unit for manually switching the flow path is provided.
  • the switching valve 7 is an example of a first switching unit according to the present invention. Note that the switching valve 7 may have a structure that can be in a state of not communicating with any of the plurality of first branch paths 416.
  • the second flow path 42 includes a second main path 425 and a plurality (three in this embodiment) of second branch paths 426.
  • One end of the second main path 425 is connected to the culture vessel unit 1 (fluid outlet 141), and the other end of the second main path 425 is connected to the switching valve 8.
  • the plurality of second branch paths 426 correspond to each of the plurality of vacuum vessel units 3, each having one end communicating with the vacuum vessel unit 3 and the other end connected to the switching valve 8.
  • the plurality of constrictions 6 are provided corresponding to the plurality of second branch paths 426, respectively.
  • the switching valve 8 selectively connects one of the plurality of vacuum vessel units 3 to the culture vessel unit 1.
  • the switching valve 8 has a valve structure capable of switching a flow path so that any one of the plurality of second branch paths 426 and the second main path 425 communicate with each other.
  • An operation unit for manually switching the flow path is provided.
  • the switching valve 8 is an example of a second switching means according to the present invention. Note that the switching valve 8 may have a structure that can be in a state of not communicating with any of the plurality of second branch paths 426. In the configuration example illustrated in FIG. 8, the plurality of first branch paths 416 and the plurality of second branch paths 426 are each separable in the middle of the flow path.
  • connection portions 47 and 48 having a luer lock connection structure, for example.
  • the perfusion culture system A20 contains culture cells and a culture solution inside a closed culture container unit 1 configured using an open culture container (container body 11), and maintains the culture state while maintaining the cell culture state. Used to make exchanges.
  • the perfusion culture system A20 of the present embodiment includes a plurality of culture solution containers 2 and a plurality of vacuum container units 3.
  • the plurality of culture solution containers 2 and the plurality of vacuum container units 3 are connected to the culture container unit 1 in advance, and the switching valves 7 and By switching the flow paths appropriately according to FIG. 8, the exchange operation of the culture solution can be performed seamlessly.
  • the perfusion culture system A20 is also useful when it is necessary to change the type of culture solution to be exchanged. That is, if different types of culture solutions are stored in the plurality of culture solution containers 2, the different types of culture solutions can be smoothly supplied to the culture vessel unit 1 simply by switching the flow path appropriately by the switching valve 7. Can be.
  • the perfusion culture system A20 is useful when changing the flow rate of the culture medium exchange during the culture medium exchange operation.
  • the flow rate of the culture solution exchange can be quickly changed only by switching the flow path appropriately by the switching valve 8. It is possible.
  • the constriction may be constituted by, for example, the microchannel chip shown in FIG.
  • the microchannel chip 6A shown in the figure is formed by, for example, attaching a hydrophilic polyethylene terephthalate (PET) film to a plate material of dimethylpolysiloxane (PDMS) in which groove-shaped microchannels 61 are formed by laser processing.
  • PET polyethylene terephthalate
  • PDMS dimethylpolysiloxane
  • the fine flow channel 61 is meandering, but by appropriately setting the width and length of the fine flow channel 61, it is possible to appropriately adjust the flow rate at the time of replacing the culture solution.
  • the culture solution container 2 may have a fixed capacity.
  • a filter for example, a hepafilter for capturing fine particles in the air is provided in the small hole.

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Le problème décrit par la présente invention est de fournir un système de culture par perfusion approprié pour économiser le travail d'un travailleur et assurer une qualité de cellule, ainsi que pour éliminer le risque de défaillance, avec une taille compacte. La solution selon l'invention porte sur un système de culture par perfusion A10 qui comprend : une unité de récipient de culture étanche 1 ayant un orifice d'entrée de fluide 131 et un orifice de sortie de fluide 141 ; un corps de stockage de solution de culture 2 apte à stocker une solution de culture ; une unité de récipient sous vide 3 dans laquelle la pression interne est ajustée à une pression négative ; un premier canal d'écoulement 41 qui est capable de communiquer avec l'intérieur de l'unité de récipient de culture 1 par l'intermédiaire de l'orifice d'entrée de fluide 131 à une extrémité de celui-ci, et qui est capable de communiquer avec l'intérieur du corps de stockage de solution de culture 2 à l'autre extrémité de celui-ci ; et un second canal d'écoulement 42 qui est capable de communiquer avec l'intérieur de l'unité de récipient de culture 1 par l'intermédiaire de l'orifice de sortie de fluide 141 à une extrémité de celui-ci, et qui est capable de communiquer avec l'intérieur de l'unité de récipient sous vide 3 à l'autre extrémité de celui-ci.
PCT/JP2018/024870 2018-06-29 2018-06-29 Système de culture par perfusion WO2020003504A1 (fr)

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JP2020110062A (ja) * 2019-01-09 2020-07-27 株式会社アイカムス・ラボ アダプタ、クローズドチャンバ、細胞培養装置及びアダプタの製造方法
CN113106019A (zh) * 2021-03-15 2021-07-13 重庆医科大学 用于椎间盘髓核组织仿生培养的静水压生物反应器
WO2023037802A1 (fr) * 2021-09-13 2023-03-16 Nok株式会社 Dispositif de culture par perfusion et système de culture par perfusion

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JP2007222063A (ja) * 2006-02-23 2007-09-06 Scimedia Ltd 培養装置及び培養方法
WO2013085020A1 (fr) * 2011-12-09 2013-06-13 Kuroiwa Yasuyuki Procédé de culture cellulaire et appareil de culture
WO2016125863A1 (fr) * 2015-02-05 2016-08-11 オリンパス株式会社 Système de culture cellulaire
JP2016536014A (ja) * 2013-10-30 2016-11-24 ジェイソン・ミクラス 三次元組織培養のためのデバイス及び方法
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WO2013085020A1 (fr) * 2011-12-09 2013-06-13 Kuroiwa Yasuyuki Procédé de culture cellulaire et appareil de culture
JP2016536014A (ja) * 2013-10-30 2016-11-24 ジェイソン・ミクラス 三次元組織培養のためのデバイス及び方法
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Publication number Priority date Publication date Assignee Title
JP2020110062A (ja) * 2019-01-09 2020-07-27 株式会社アイカムス・ラボ アダプタ、クローズドチャンバ、細胞培養装置及びアダプタの製造方法
JP7262042B2 (ja) 2019-01-09 2023-04-21 株式会社アイカムス・ラボ アダプタ、クローズドチャンバ、細胞培養装置及びアダプタの製造方法
CN113106019A (zh) * 2021-03-15 2021-07-13 重庆医科大学 用于椎间盘髓核组织仿生培养的静水压生物反应器
WO2023037802A1 (fr) * 2021-09-13 2023-03-16 Nok株式会社 Dispositif de culture par perfusion et système de culture par perfusion

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