WO2023037802A1 - Dispositif de culture par perfusion et système de culture par perfusion - Google Patents

Dispositif de culture par perfusion et système de culture par perfusion Download PDF

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Publication number
WO2023037802A1
WO2023037802A1 PCT/JP2022/029906 JP2022029906W WO2023037802A1 WO 2023037802 A1 WO2023037802 A1 WO 2023037802A1 JP 2022029906 W JP2022029906 W JP 2022029906W WO 2023037802 A1 WO2023037802 A1 WO 2023037802A1
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Prior art keywords
perfusion culture
channel
wall portion
closed space
bottom plate
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Application number
PCT/JP2022/029906
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English (en)
Japanese (ja)
Inventor
諒 二嶋
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Nok株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nok株式会社 filed Critical Nok株式会社
Priority to JP2023546836A priority Critical patent/JPWO2023037802A1/ja
Publication of WO2023037802A1 publication Critical patent/WO2023037802A1/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 perfusion culture device and a perfusion culture system.
  • a perfusion culture system that includes a closed culture vessel unit and first and second flow paths communicating with the culture vessel unit.
  • a perfusion culture system described in Patent Document 1, by using a pump or the like, a new culture medium is supplied to the culture vessel from one of the two channels, and the old culture medium is removed from the other channel, which is automated. It is
  • the present invention has been made in view of the above-mentioned problems, and its purpose is to provide a perfusion culture apparatus and a perfusion culture system that can improve the efficiency of liquid feeding.
  • a perfusion culture apparatus is a perfusion culture apparatus that is attached to an opening of a culture vessel that includes a bottom plate and a peripheral wall that is erected from the bottom plate, A closed portion that closes an opening to form a closed space in the culture vessel, an introduction channel and an outlet channel that penetrate the closed portion and communicate with the closed space, and the introduction channel and the outlet channel. and a bubble flow path that is formed independently of and penetrates the closed portion and communicates with the closed space, and the bubble flow path includes a filter that allows gas to pass through and regulates the passage of liquid. is provided.
  • At least a portion of the closing portion disposed inside the peripheral wall portion is integrally molded with an elastic member, and the surface of the closing portion facing the bottom plate portion is: The inlet channel and the outlet channel open to the closed space at positions along the inner side of the peripheral wall.
  • the blocking portion includes a plate portion facing the bottom plate portion, and a wall portion erected from the plate portion and arranged along the inner side of the peripheral wall portion. , wherein the introduction channel, the outlet channel, and the air bubble channel are formed to pass through the wall portion.
  • a lip portion extending in the circumferential direction and protruding toward the peripheral wall portion is provided on the outer peripheral surface of the portion of the closing portion that is arranged inside the peripheral wall portion. is provided.
  • the blocking portion includes a plate portion facing the bottom plate portion, and a wall portion erected from the plate portion and arranged along the inner side of the peripheral wall portion. and a bead portion that opens to the side opposite to the bottom plate portion and that connects the plate portion and the wall portion to the opening side.
  • At least one of the introduction channel, the outlet channel, and the air bubble channel has an end portion on the closed space side that, as the distance from the closed space increases, A tapered portion is formed to reduce the inner diameter.
  • a perfusion culture system of the present invention includes any of the perfusion culture devices described above and the culture vessel.
  • the perfusion culture apparatus and the perfusion culture system according to the present invention it is possible to improve the liquid feeding efficiency.
  • FIG. 1 is a perspective view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a cross-sectional view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a partial cross-sectional view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a cross-sectional view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is an enlarged sectional view showing a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a perspective view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a cross-sectional view of a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 1 is a partial cross
  • FIG. 1 is an enlarged sectional view showing a perfusion culture system equipped with a perfusion culture device according to an embodiment of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 10 is an enlarged cross-sectional view showing a perfusion culture system equipped with a perfusion culture apparatus according to a modification of the present invention
  • FIG. 11 is a perspective view showing a connecting member of a perfusion culture system having a perfusion culture device according to a modification of the present invention
  • FIG. 1 is a perspective view of a perfusion culture system 200 having a perfusion culture apparatus 1 according to an embodiment of the present invention
  • FIG. 2 is a cross-sectional view of the perfusion culture system 200
  • FIG. 3 is a perfusion culture system
  • 4 is a cross-sectional view of the perfusion culture system 200
  • FIGS. 5 and 6 are cross-sectional views showing the perfusion culture system 200 on an enlarged scale.
  • the perfusion culture apparatus 1 includes a culture vessel 100 having a bottom plate portion 101 and a peripheral wall portion 102 erected from the bottom plate portion 101.
  • a perfusion culture apparatus 1 to be attached comprising: a blocking portion 10 that blocks an opening 103 to form a closed space A1 in a culture container 100; an introduction channel 6 that penetrates the blocking portion 10 and communicates with the closed space A1; an outlet channel 7; and a bubble channel 8 formed independently of the inlet channel 6 and the outlet channel 7, penetrating the closed part 10 and communicating with the closed space A1, wherein the bubble channel 8 is provided with a filter 9 that allows passage of gas and restricts passage of liquid.
  • the perfusion culture apparatus 1 will be specifically described below.
  • the culture vessel 100 includes a disk-shaped bottom plate portion 101 and a cylindrical peripheral wall portion 102 erected from the outer peripheral edge of the bottom plate portion 101, and is a container for containing a culture solution for culturing cells and the like. For example, a petri dish or a dish may be used. Also, the culture vessel 100 is preferably transparent as a whole.
  • the culture vessel 100 has a bottom plate portion 101 and a peripheral wall portion 102 and is formed in a cylindrical shape with a bottom, thereby having an opening portion 103 .
  • the direction orthogonal to the bottom plate portion 101 (the direction in which the peripheral wall portion 102 extends) is simply referred to as the “vertical direction”, and in this vertical direction, the opening side of the culture vessel 100 is simply referred to as the “upper side”, and vice versa. The side is sometimes simply referred to as the "lower side”. It should be noted that the vertical direction here is for convenience and does not have to coincide with the vertical direction in actual use.
  • the perfusion culture device 1 is integrally molded as a whole with a transparent elastic member.
  • the transparent elastic member include silicone rubber such as polydimethylsiloxane (PDMS).
  • the perfusion culture apparatus 1 includes a plate portion 2 provided parallel to and facing a bottom plate portion 101, a wall portion 3 erected from the plate portion 2 and arranged along the inner side of the peripheral wall portion 102, and an annular and the flange portion 4, and is open to the upper side (opposite side to the bottom plate portion 101). The entire perfusion culture apparatus 1 closes the opening 103 to form the blocking section 10 .
  • the plate portion 2 is formed in a disc shape like the bottom plate portion 101 and has an outer diameter approximately equal to the inner diameter of the peripheral wall portion 102 .
  • the wall portion 3 is provided along the outer peripheral edge of the plate portion 2 , is formed in a cylindrical shape like the peripheral wall portion 102 , and has an outer diameter approximately equal to the inner diameter of the peripheral wall portion 102 .
  • the flange portion 4 extends from the upper end of the wall portion 3 (the end portion opposite to the bottom plate portion 101 ) toward the outer peripheral side, and has an outer diameter larger than the inner diameter of the peripheral wall portion 102 .
  • the corner between the plate portion 2 and the wall portion 3 is included in the plate portion 2, and the corner between the flange portion 4 and the wall portion 3 is the flange portion 4 shall be included in
  • each part of the perfusion culture apparatus 1 has the above-described dimensional relationship, the plate part 2 and the wall part 3 are inserted into the opening 103 of the culture vessel 100, and the flange part 4 restricts excessive insertion. It's becoming At this time, the perfusion culture apparatus 1 is press-fitted into the culture vessel 100, and the outer diameters of the plate portion 2 and the wall portion 3 are adjusted to the peripheral wall portion 102 so that the plate portion 2 and the wall portion 3 can be in close contact with the peripheral wall portion 102. is preferably slightly larger than the inner diameter of the In this manner, the plate portion 2 and the wall portion 3 are arranged inside the peripheral wall portion 102 to constitute the inner portion 5 .
  • the perfusion culture apparatus 1 includes an introduction channel 6 that penetrates (passes) the plate portion 2, the wall portion 3, and the flange portion 4 along the vertical direction (that is, penetrates the blocking portion 10), an outlet channel 7, and bubbles.
  • a flow path 8 is formed.
  • one introduction channel 6 and one discharge channel 7 are provided, and two bubble channels 8 are provided.
  • the inlet channel 6 and the outlet channel 7 are arranged so as to face each other in the radial direction, the bubble channel 8 is arranged so as to be adjacent to the inlet channel 6 and the outlet channel 7, and the two bubble channels 8 are arranged They are diametrically opposed.
  • Each flow path 6, 7, 8 is opened at a lower surface 21 of the plate portion 2 facing the bottom plate portion 101, and an upper surface 41 of the flange portion 4 facing away from the bottom plate portion 101. Open at
  • One end of the tube is connected to the introduction channel 6 using an adhesive or the like at the opening of the upper surface 41 of the flange portion 4 .
  • the other end of this tube is connected to a container containing a new culture medium.
  • One end of the tube is connected to the lead-out flow path 7 using an adhesive or the like at the opening of the upper surface 41 of the flange portion 4 .
  • the other end of this tube is connected to a collecting section where old culture medium is collected.
  • a liquid feeding unit such as a pump is provided for these tubes.
  • a filter 9 is provided on the upper surface 41 of the flange portion 4 so as to cover the opening of the air bubble flow path 8 .
  • the filter 9 is a membrane that allows passage of gas (eg, air) and restricts passage of liquid.
  • gas eg, air
  • An example of a membrane that is permeable to air but impermeable to liquids is a porous membrane formed from a hydrophobic resin (eg, PTFE (polytetrafluoroethylene), PFA (perfluoroalkoxyalkane)).
  • PTFE polytetrafluoroethylene
  • PFA perfluoroalkoxyalkane
  • the position where the filter is provided is not limited to the above, and a configuration in which the filter is packed in the air bubble flow path 8 may be employed.
  • the filter 9 has a dustproof property so that foreign matter can be prevented from entering the air bubble flow path 8 from the external space.
  • a perfusion culture system 200 is configured by attaching the perfusion culture apparatus 1 as described above to the culture vessel 100 .
  • a closed space A1 surrounded by the bottom plate portion 101, the peripheral wall portion 102, and the plate portion 2 is formed.
  • Each flow path 6, 7, 8 extends linearly in the vertical direction, and as shown in FIG. is open.
  • the perfusion culture system 200 is provided with a control section, and the control section drives the liquid feeding section at a predetermined timing to feed a predetermined amount of culture solution. That is, a new culture solution is sent into the closed space A1, and at the same time, an old culture solution is discharged to replace the culture solution.
  • gas may be generated or mixed in the culture solution. Such gas can pass through the bubble channel 8 together with the culture medium.
  • the culture solution and air bubbles reach the upper end of the air bubble channel 8, only the air bubbles can pass through the filter 9 and are removed from the closed space A1. At this time, leakage of the culture medium is suppressed, and airtightness of the culture space formed in the closed space A1 is ensured.
  • the bubble flow path 8 is provided, and the filter 9 that allows gas to pass through and regulates the passage of liquid is provided. , air bubbles can be removed, and the liquid transfer efficiency can be improved.
  • the introduction channel 6 and the outlet channel 7 are open to the closed space A1 at positions along the inner side of the peripheral wall portion 102 on the lower surface 21, the closed space A1 near the peripheral wall portion 102 is opened.
  • the culture solution located at the corner can also be exchanged efficiently.
  • the introduction channel 6, the outlet channel 7, and the bubble channel 8 are formed so as to pass through the wall portion 3 along the inner side of the peripheral wall portion 102, the peripheral wall portion 102 of the closed space A1 It is also possible to efficiently exchange the culture medium located at the nearby corners.
  • the flow path is formed so as to pass through only the plate-shaped portion, when the tube is connected to the introduction flow path 6 and the discharge flow path 7, deformation at the connection portion is suppressed. It's easy to do.
  • the bubble flow path 8 is provided adjacent to the outlet flow path 7 , when the culture solution flows toward the outlet flow path 7 , air bubbles tend to flow toward the bubble flow path 8 .
  • the two bubble flow paths 8 are arranged so as to face each other in the radial direction, when the bottom plate portion 101 is inclined with respect to the horizontal direction and the outlet flow path 7 side is lower, the introduction flow path 6 Air bubbles are likely to face the air bubble flow path 8 adjacent to the .
  • the present invention is not limited to the above embodiments, but includes other configurations and the like that can achieve the object of the present invention, and the following modifications and the like are also included in the present invention.
  • the entire perfusion culture apparatus 1 is integrally molded with a transparent elastic member, but the perfusion culture apparatus may be configured with a plurality of members. It is sufficient that at least a part thereof is made of an elastic member so that it can be attached to the culture vessel. Also, the perfusion culture device may be partially or wholly opaque.
  • the inlet channel 6, the outlet channel 7, and the air bubble channel 8 are not limited to have constant inner diameters, and these channels include, for example, the closed space A1 side ( Tapered portions 61 , 62 , 81 , 82 may be formed at the end portion of the lower side in the drawing so that the inner diameter becomes smaller as the distance from the closed space A ⁇ b>1 increases.
  • the tapered portions 61 and 81 in FIG. 7 have curved cross sections (convex downward), and the tapered portions 62 and 82 in FIG. 8 have straight cross sections.
  • the inlet channel 6 and the bubble channel 8 are formed with tapered portions, but the outlet channel 7 may also be formed with a similar tapered portion.
  • the taper portion may be formed in all of the above, or may be formed only in a part thereof.
  • the culture solution can easily pass between each of the channels 6-8 and the closed space A1.
  • the entire outer peripheral surface of the blocking portion 10 is not limited to contacting the peripheral wall portion 102.
  • the entire outer peripheral surface of the blocking portion 10 is not limited to contacting the peripheral wall portion 102.
  • FIGS. 2 and wall 3 are provided with lips 22 and 32 extending along the circumferential direction and protruding toward the peripheral wall 102, and only the lips 22 and 32 contact the peripheral wall 102.
  • the lip portion 22 is provided only at the position corresponding to the plate portion 2, and in the example shown in FIG. 32 are provided.
  • the contact pressure of the closed portion 10 against the peripheral wall portion 102 can be improved, and the sealing performance of the closed space A1 can be improved.
  • the perfusion culture apparatus 1 is provided with bead portions 300 and 400 connecting the plate portion 2 and the wall portion 3 on the opening side (opposite side to the bottom plate portion 101).
  • the bead portions 300 and 400 are formed so as to extend over the upper surface 23 of the plate portion 2 and the inner peripheral surface 33 of the wall portion 3, and are formed over the entire circumferential direction.
  • the bead portion 300 shown in FIG. 11 bulges upward and has a curved upper surface
  • the bead portion 400 shown in FIG. 12 has a linear upper surface.
  • the rigidity of the perfusion culture apparatus 1 can be improved. For example, it is suppressed that the upper end side of the wall portion 3 deforms so as to reduce its diameter. By improving the rigidity and suppressing deformation in this manner, the sealing performance of the closed space A1 can be improved.
  • the tubes are directly connected to the channels 6 and 7 of the perfusion culture apparatus 1. 6, 7 and a tube may be connected.
  • One end portion 501 of the connection member 500 is inserted into the openings of the flow paths 6 and 7 on the upper surface 41 of the flange portion 4, and the other end portion 502 is inserted into the tube.
  • two air bubble channels 8 are formed in the perfusion culture apparatus 1 and arranged so as to face each other in the radial direction. Not limited.
  • one air bubble channel 8 may be provided.
  • the air bubble channel 8 is preferably arranged in the vicinity of the outlet channel 7, but may be arranged at another position. Also, three or more air bubble flow paths 8 may be provided.
  • the introduction channel 6, the outlet channel 7, and the air bubble channel 8 are formed so as to pass through the wall portion 3.
  • the opening positions of the flow paths 6 to 8 with respect to the closed space A1 are not limited to positions along the inner side of the peripheral wall portion 102, either. That is, the channels 6 to 8 may be arranged so that the culture medium is sufficiently exchanged. position.
  • the present invention is not limited to the perfusion culture apparatus according to the above-described embodiments of the present invention. include. Moreover, each configuration may be selectively combined as appropriate so as to achieve at least part of the above-described problems and effects. For example, the shape, material, arrangement, size, etc. of each component in the above embodiment may be changed as appropriate according to the specific usage of the present invention.
  • SYMBOLS 1 Perfusion culture apparatus, 2... Plate part, 21... Lower surface, 22... Lip part, 23... Upper surface, 3... Wall part, 32... Lip part, 4... Flange part, 41... Upper surface, 5... Inside arrangement part, 6 Introductory channel, 7 Outlet channel, 8 Air bubble channel, 9 Filter, 10 Blocking part, 100 Culture vessel, 101 Bottom plate part, 102 Peripheral wall part, 103 Opening, 200 Perfusion culture System 300, 400 Bead portion 500 Connecting member 501 One end portion 502 Other end portion A1 Closed space

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  • Bioinformatics & Cheminformatics (AREA)
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Abstract

La présente invention concerne un dispositif de culture par perfusion et un système de culture par perfusion capables d'améliorer l'efficacité de l'alimentation en liquide.　Dispositif de culture par perfusion (1) fixé à une ouverture (103) d'un récipient de culture (100) comportant une plaque inférieure (101) et une paroi périphérique (102) érigée à partir de la plaque inférieure (101), le dispositif de culture par perfusion (1) comprenant : une partie fermante (10) qui ferme l'ouverture (103) pour définir un espace fermé (A1) dans le récipient de culture (100) ; un canal d'entrée (6) et un canal de sortie (7) traversant la partie fermante (10) et communiquant avec l'espace fermé (A1) ; et un chemin d'écoulement à bulles (8) formé indépendamment du canal d'entrée (6) et du canal de sortie (7), traversant la partie fermante (10) et communiquant avec l'espace fermé (A1), un filtre (9) permettant le passage de gaz et limitant le passage de liquide étant prévu dans le chemin d'écoulement à bulles (8).
PCT/JP2022/029906 2021-09-13 2022-08-04 Dispositif de culture par perfusion et système de culture par perfusion WO2023037802A1 (fr)

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JP2023546836A JPWO2023037802A1 (fr) 2021-09-13 2022-08-04

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JP2021148881 2021-09-13
JP2021-148881 2021-09-13

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04506149A (ja) * 1989-05-05 1992-10-29 セントコール，インコーポレイテッド 改良された細胞培養系
JP2005514058A (ja) * 2001-12-21 2005-05-19 オーガノジェネシス インコーポレーテッド 細胞培養及び臓器補助用の可変容量チャンバ
JP2007202542A (ja) * 2006-02-02 2007-08-16 Atoo Kk 細胞培養容器
WO2013086486A1 (fr) * 2011-12-09 2013-06-13 President And Fellows Of Harvard College Systèmes microphysiologiques d'organes-sur-puce humains intégrés
US20150298123A1 (en) * 2010-10-07 2015-10-22 Vanderbilt University Organ on chip integration and applications of the same
WO2020003504A1 (fr) * 2018-06-29 2020-01-02 株式会社サンプラテック Système de culture par perfusion

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04506149A (ja) * 1989-05-05 1992-10-29 セントコール，インコーポレイテッド 改良された細胞培養系
JP2005514058A (ja) * 2001-12-21 2005-05-19 オーガノジェネシス インコーポレーテッド 細胞培養及び臓器補助用の可変容量チャンバ
JP2007202542A (ja) * 2006-02-02 2007-08-16 Atoo Kk 細胞培養容器
US20150298123A1 (en) * 2010-10-07 2015-10-22 Vanderbilt University Organ on chip integration and applications of the same
WO2013086486A1 (fr) * 2011-12-09 2013-06-13 President And Fellows Of Harvard College Systèmes microphysiologiques d'organes-sur-puce humains intégrés
WO2020003504A1 (fr) * 2018-06-29 2020-01-02 株式会社サンプラテック Système de culture par perfusion

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