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WO2022190261A1 - Récipient de culture cellulaire - Google Patents

Récipient de culture cellulaire Download PDF

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Publication number
WO2022190261A1
WO2022190261A1 PCT/JP2021/009546 JP2021009546W WO2022190261A1 WO 2022190261 A1 WO2022190261 A1 WO 2022190261A1 JP 2021009546 W JP2021009546 W JP 2021009546W WO 2022190261 A1 WO2022190261 A1 WO 2022190261A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
culture vessel
container body
tubular portion
bottom portion
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2021/009546
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English (en)
Japanese (ja)
Inventor
学司 加藤
順一 桑原
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SANPLATEC Corp Ltd
Original Assignee
SANPLATEC Corp Ltd
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 SANPLATEC Corp Ltd filed Critical SANPLATEC Corp Ltd
Priority to PCT/JP2021/009546 priority Critical patent/WO2022190261A1/fr
Priority to JP2023504964A priority patent/JP7765842B2/ja
Publication of WO2022190261A1 publication Critical patent/WO2022190261A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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 disclosure relates to a cell culture vessel for culturing cells, and in particular, to a cell culture vessel used for perfusion culture that automatically supplies culture medium at a constant low speed and discharges the same amount of culture medium at the same time.
  • culture vessels such as dishes (petri dishes), well plates, and flasks are widely used. Even if the dishes and well plates had lids, there was a gap just by putting them on, and it was impossible to seal them. Flasks can be hermetically sealed, but all of these conventional culture vessels require the opening and closing of lids for operations such as exchanging the culture solution. Therefore, these culture vessels are highly versatile for use in cell culture for research purposes. There was a problem that it was difficult to equalize the quality of cultured cells.
  • a culture vessel used in an automatic culture apparatus is provided with, for example, a pair of ports. These ports are provided, for example, in the lid of the culture vessel and extend downward from the lid to be arranged inside the culture vessel.
  • a new culture solution is supplied to the culture container through one port, and the culture solution inside the culture container is discharged to the outside through the other port.
  • the amount of culture medium supplied from one port and the amount of culture medium discharged from the other port are not exactly the same, the liquid in the container may dry up or overflow during long-term cultivation. Become.
  • one pump is used for one container, and the culture solution is supplied from one port under positive pressure and discharged from the other port by overflowing, or A method in which the culture medium is discharged from the port under negative pressure and the culture medium is drawn in from the other port is effective. If this is done all at once, turbulence will occur and the cells will be damaged. Therefore, perfusion culture is performed in which the culture solution is automatically supplied at a constant low speed and the same amount of culture solution is discharged at the same time. In this perfusion culture, in order to realize smooth liquid flow, it is important to have a closed system and to fill the entire interior of the culture vessel with the culture medium without creating a gas layer or air bubbles.
  • the present disclosure has been conceived under such circumstances, and a main object of the present disclosure is to provide a cell culture vessel suitable for improving the inconvenience of performing perfusion culture in a closed system. .
  • a cell culture vessel includes a vessel body having an opening at one end in a first direction and having a first cylindrical portion extending in the first direction, and a plug member capable of closing the opening. and, the plug member includes a second cylindrical portion that abuts and seals the inner peripheral surface of the first cylindrical portion, and a diameter from the one side end of the second cylindrical portion in the first direction. a first flange portion that extends outward in the direction and closes the one side end of the first tubular portion in the first direction; a plate-like first bottom part that extends in the direction of the first direction and closes the inside of the second cylindrical part when viewed in the first direction, and the first bottom part has the one side end in the first direction that extends to the outside.
  • a plurality of ports each having a flow path that opens to the first direction and that the other side end in the first direction opens in the inner space of the container body, and the first bottom portion is provided in the first direction a ceiling surface facing the other side, a ceiling recess recessed from the ceiling surface toward the one side in the first direction is formed in the first bottom portion, and the ceiling recess is one of the plurality of ports; Communicate with at least one of the channels.
  • the ceiling recess communicates with the outer peripheral edge of the first bottom.
  • annular top plate portion a third tubular portion extending from the outer peripheral edge of the top plate portion in the thickness direction of the top plate portion and fitted onto the first tubular portion, At least one of the container body and the outer lid has the first tubular portion and the third flange portion in contact with the first tubular portion. Locking means are provided to prevent relative movement with the tubular portion.
  • the plug member is made of a soft material.
  • it further comprises a cover member made of a hard material and overlapping and abutting on the plug member.
  • the cover member includes at least a second flange portion covering the first flange portion, a fourth tubular portion covering an inner peripheral surface of the second tubular portion, and the first bottom portion. a second bottom covering a portion.
  • the port has an extension extending from the first bottom to the one side in the first direction, and the cover member is in contact with the outer peripheral surface of the extension.
  • a fifth tubular portion surrounding the outer peripheral surface is included.
  • the first bottom portion includes a thin portion having a relatively smaller thickness than other portions and having gas permeability.
  • the first bottom portion has a thickness relatively smaller than that of other portions and includes a thin portion having gas permeability
  • the second bottom portion includes A through hole is formed to penetrate through, and the through hole overlaps with the thin portion when viewed in the first direction.
  • the tip of the extending portion is provided with a protruding portion that protrudes outward from the outer peripheral surface and can come into contact with the one side end of the fifth tubular portion in the first direction. is provided.
  • the flow path comprises a first flow path open to the outside and a second flow path open to the inner space of the container body and connected to the ceiling recess.
  • the channel and the second channel are spaced apart from each other in the first direction and overlap when viewed in the first direction.
  • FIG. 2 is an exploded perspective view of the cell culture vessel shown in FIG. 1;
  • FIG. 2 is a plan view of the cell culture vessel shown in FIG. 1;
  • 4 is an enlarged cross-sectional view taken along line IV-IV of FIG. 3;
  • FIG. 4 is an enlarged cross-sectional view taken along line VV of FIG. 3;
  • FIG. 4 is a bottom view of a plug member that constitutes the cell culture vessel according to the first embodiment;
  • FIG. 5 is a cross-sectional view, similar to FIG. 4, showing the state of use of the cell culture vessel according to the first embodiment;
  • FIG. 5 is a cross-sectional view, similar to FIG.
  • FIG. 4 showing the state of use of the cell culture vessel according to the first embodiment
  • FIG. 5 is a cross-sectional view, similar to FIG. 4, showing the state of use of the cell culture vessel according to the first embodiment
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a first modified example of the first embodiment
  • FIG. 11 is a bottom view of a plug member that constitutes a cell culture vessel according to a first modified example of the first embodiment
  • FIG. 11 is a bottom view showing another example of the plug member
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a second modified example of the first embodiment
  • FIG. 5 is a cross-sectional view similar to FIG.
  • FIG. 4 showing a cell culture vessel according to a third modified example of the first embodiment
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a fourth modified example of the first embodiment
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a second embodiment
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a third embodiment
  • FIG. 5 is a cross-sectional view similar to FIG. 4 showing a cell culture vessel according to a fourth embodiment
  • an entity A overlaps an entity B when viewed in a certain direction means, unless otherwise specified, “an entity A overlaps all of an entity B” and “an entity A overlaps an entity B.” "A overlaps part of something B”.
  • FIGS. 1 and 3 to 5 show an assembled state in which the container body 1, the plug member 2, the cover member 3 and the outer lid 4 are combined.
  • FIG. 2 is an exploded perspective view of constituent members of the cell culture vessel A1.
  • the container body 1 is provided with a first cylindrical portion 11 and a container bottom portion 12, and has a container shape having an opening 110 at an upper end 114 (one side end in the first direction) of the first cylindrical portion 11. It is
  • the first tubular portion 11 has a substantially cylindrical shape and extends in the vertical direction (first direction).
  • a male screw 111 is formed on the upper outer peripheral surface of the first tubular portion 11 .
  • the container bottom portion 12 is connected to the vicinity of the lower end of the first tubular portion 11 (near the other side end in the first direction) and closes the inside of the first tubular portion 11 .
  • a slit 112 is formed at an appropriate location on the lower end of the first tubular portion 11 .
  • an extension piece 113 is provided at a suitable position on the lower end of the first tubular portion 11 .
  • the extension piece 113 is a portion that is connected to the first cylindrical portion 11 and extends radially outward.
  • the extension piece 113 is provided at a position different from the slit 112 in the circumferential direction of the first tubular portion 11 .
  • the container body 1 is made of, for example, a translucent or transparent plastic material.
  • plastic material include, but are not limited to, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other transparent materials.
  • the upper surface of the container bottom 12 is a flat cell culture surface for culturing cells.
  • This cell culture surface (the upper surface of the container bottom portion 12) is appropriately subjected to a surface treatment for improving cell adhesiveness, if necessary.
  • the surface treatment include hydrophilic treatment such as corona discharge treatment and plasma treatment.
  • the plug member 2 is for closing the opening 110 of the container body 1 and sealing the container body 1 .
  • the plug member 2 is, for example, a molded rubber product, and includes a second tubular portion 21, a first flange portion 22, a first bottom portion 23 and a plurality of ports 24, as shown in FIGS. consists of
  • the second cylindrical portion 21 has a substantially cylindrical shape, and contacts and seals the inner peripheral surface of the first cylindrical portion 11 .
  • a pair of annular protrusions 211 are formed on the outer peripheral portion of the second tubular portion 21 at intervals in the vertical direction (first direction).
  • the outer diameter dimension of the annular protrusion 211 in the natural state is slightly larger than the inner diameter dimension of the first cylindrical portion 11, and when the plug member 2 is attached to the container body 1, the second cylindrical portion 21 is It is fitted into the first cylindrical portion 11 while being compressed radially inward. Then, the annular protrusion 211 is in close contact with the inner peripheral surface of the first cylindrical portion 11 to seal it.
  • the second tubular portion 21 may be configured without the annular projection 211 . If the annular projection 211 is not provided, the outer peripheral surface of the second tubular portion 21 surface-contacts with the inner peripheral surface of the first tubular portion 11 for sealing.
  • the first flange portion 22 has a substantially annular shape and extends radially outward from the upper end (one side end in the first direction) of the second cylindrical portion 21 .
  • the first flange portion 22 overlaps with the first tubular portion 11 when viewed in the thickness direction of the first flange portion 22 and closes the upper end of the first tubular portion 11 .
  • the first flange portion 22 has an appropriate thickness and has an appropriate elastic restoring force against a load in the vertical direction (first direction).
  • the thickness of the first flange portion 22 is, for example, about 1 to 3 mm.
  • the first bottom portion 23 extends radially inward from the lower end of the second tubular portion 21 (the other end in the first direction), and closes the inside of the second tubular portion 21 when viewed in the vertical direction.
  • the first bottom portion 23 is plate-shaped and has a ceiling surface 231 .
  • the ceiling surface 231 is a flat surface facing downward (the other side in the first direction).
  • a ceiling concave portion 232 is formed in the first bottom portion 23 .
  • the ceiling recess 232 is a portion recessed upward (one side in the first direction) from the ceiling surface 231 .
  • the ceiling recessed portion 232 is provided near the outer periphery of the first bottom portion 23 . Although the details will be described later, the ceiling recess 232 is arranged corresponding to the port 24 .
  • the first bottom portion 23 has a thin portion 238 .
  • the thin portion 238 is a portion that is thinner than other portions.
  • the thin portion 238 is arranged in the center of the first bottom portion 23 and has a substantially circular shape when viewed in the vertical direction (first direction).
  • the top surface of the first bottom portion 23 is recessed at the central portion thereof, and the recessed portion of the top surface of the first bottom portion 23 is the thin portion 238 .
  • FIG. 6 the boundary between the thin portion 238 and a portion surrounding the thin portion 238 when viewed in the thickness direction (first direction) (hereinafter referred to as "peripheral thick portion 235" is indicated by a virtual line.
  • the thin portion 238 has gas permeability. Also in this embodiment, thinned portion 238 is transparent.
  • the thickness of the thin portion 238 is, for example, approximately 0.2 to 0.3 mm.
  • the thickness of the outer peripheral thick portion 235 surrounding the thin portion 238 is about the same as the thickness of the first flange portion 22, for example, about 1 to 3 mm.
  • a plurality of ports 24 are provided on the first bottom portion 23 and are integrally formed at appropriate locations on the first bottom portion 23 .
  • the first bottom portion 23 is provided with a pair of ports 24 .
  • the pair of ports 24 are provided near the outer periphery of the first bottom portion 23 and are arranged on opposite sides of the center of the first bottom portion 23 when viewed in the vertical direction (first direction).
  • Each port 24 is provided in the outer thick portion 235 .
  • the port 24 is for supplying the culture solution to the inside of the container body 1 or discharging the culture solution inside the container body 1 to the outside, and as shown in FIG. have.
  • the extending portion 241 has a cylindrical shape extending upward (one side in the first direction) from the first bottom portion 23 and has an outer peripheral surface 241a having a circular cross section.
  • a projection 245 is provided at the tip of the extension 241 and projects outward from the outer peripheral surface 241a.
  • the flow path 242 has an upper end (one side end in the first direction) open to the outside and a lower end (hand side end in the first direction) open to the inner space of the container body 1 so that the outside and the container body are open. It is a communication passage leading to the inner space of 1.
  • the channel 242 has a circular cross section and is formed in the extension 241 .
  • the ceiling recessed portion 232 overlaps the port 24 when viewed in the vertical direction (first direction) and is connected to the flow path 242 .
  • the ceiling recessed portion 232 has a conical shape, and is formed such that the area of the cross section increases downward from the position where it is connected to the flow path 242 .
  • the ceiling recess 232 is formed across the first bottom portion 23 and the port 24 (extending portion 241).
  • the plug member 2 configured as described above is made of a soft material having flexibility and elasticity.
  • the material forming the plug member 2 include silicone rubber and elastomer resin.
  • the material of the plug member 2 is more preferably non-cytotoxic and biocompatible medical silicone rubber.
  • the hardness of the plug member 2 it is preferable that the rubber hardness is about 20 to 40 degrees, for example.
  • the cover member 3 overlaps the plug member 2 and contacts the plug member 2 .
  • the cover member 3 includes a second flange portion 31 , a fourth tubular portion 32 , a second bottom portion 33 and a fifth tubular portion 34 .
  • the second flange portion 31 covers the first flange portion 22 from above and is in contact with the upper surface of the first flange portion 22 .
  • the fourth tubular portion 32 is connected to the inner peripheral edge of the second flange portion 31 .
  • the fourth tubular portion 32 has a substantially cylindrical shape and extends downward (the other side in the first direction) from the inner peripheral edge of the second flange portion 31 .
  • the fourth tubular portion 32 covers the inner peripheral surface of the second tubular portion 21 and is in contact with the inner peripheral surface of the second tubular portion 21 .
  • the second bottom portion 33 is connected to the lower end of the fourth tubular portion 32 (the other end in the first direction) and extends radially inward from the lower end of the fourth tubular portion 32 .
  • the second bottom portion 33 covers the first bottom portion 23 from above and is in contact with the upper surface of the first bottom portion 23 .
  • the central portion of the second bottom portion 33 is recessed downward (on the other side in the first direction) corresponding to the location where the thin portion 238 is formed.
  • a through hole 331 is formed in the second bottom portion 33 .
  • the through hole 331 penetrates the second bottom portion 33 in the thickness direction and overlaps the thin portion 238 of the plug member 2 when viewed in the vertical direction (first direction).
  • a plurality of through holes 331 are formed in the second bottom portion 33 . As shown in FIG. 3 and the like, the plurality of through holes 331 are appropriately distributed when viewed in the vertical direction.
  • the fifth tubular portion 34 is provided on the second bottom portion 33 and extends upward (one side in the first direction) from a suitable location on the second bottom portion 33 .
  • the second bottom portion 33 is provided with a pair of fifth cylindrical portions 34 .
  • the pair of fifth cylindrical portions 34 are arranged corresponding to the pair of ports 24, respectively.
  • each fifth tubular portion 34 is shaped to be connected to a portion of the fourth tubular portion 32 .
  • the port 24 (extending portion 241) is fitted in each of the pair of fifth cylindrical portions 34.
  • the inner diameter dimension of the fifth tubular portion 34 is slightly smaller than the outer diameter dimension of the extending portion 241 in the natural state.
  • Each extending portion 241 is press-fitted into the fifth tubular portion 34 .
  • the fifth tubular portion 34 is in close contact with (contacts with) the outer peripheral surface 241a of the extending portion 241 to surround the outer peripheral surface 241a.
  • the fifth cylindrical portion 34 overlaps the protrusion 245 provided at the tip of the port 24. there is The projecting portion 245 is adjacent above the fifth tubular portion 34 . As a result, the protrusion 245 can come into contact with the upper end of the fifth tubular portion 34 .
  • the cover member 3 is made of a hard material, such as a translucent or transparent plastic material.
  • a hard material such as a translucent or transparent plastic material.
  • the plastic material include, but are not limited to, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other transparent materials.
  • the outer lid 4 has a top plate portion 41 and a third tubular portion 42 .
  • the top plate portion 41 has a substantially annular shape and is a portion that sandwiches the second flange portion 31 of the cover member 3 and the upper end 114 of the first cylindrical portion 11 of the container body 1 .
  • a through hole 411 is formed radially inside the top plate portion 41 .
  • the through hole 411 is surrounded by the top plate portion 41 when viewed in the thickness direction of the top plate portion 41 .
  • the through hole 411 overlaps the thin portion 238 of the plug member 2 and the plurality of through holes 331 of the cover member 3 when viewed in the thickness direction (vertical direction) of the top plate portion 41 .
  • the third tubular portion 42 extends from the outer peripheral edge of the top plate portion 41 in the thickness direction (downward in the figure) of the top plate portion 41 and has a substantially cylindrical shape.
  • a female screw 421 is formed on the inner peripheral surface of the third tubular portion 42, and the female screw 421 can be screwed into the male screw 111 of the container body 1 (first tubular portion 11).
  • the top plate portion 41 is provided with a convex portion 412 .
  • the convex portion 412 protrudes upward (one side in the first direction) from the main surface 41a of the top plate portion 41 facing the thickness direction.
  • a plurality of convex portions 412 are provided separately from each other.
  • a plane P ⁇ b>1 formed by the tips 412 a of these convex portions 412 is substantially parallel to the main surface 41 a of the top plate portion 41 .
  • substantially parallel is designed so that the plane P1 and the main surface 41a are parallel, but it also includes the case where there is some variation in parallelism due to dimensional tolerance or the like.
  • a plane P1 formed by the tips 412a of the plurality of convex portions 412 is located at the highest position in the cell culture container A1.
  • the convex portions 412 are provided in the shape of concentric arcs when viewed in the up-down direction, and gaps are formed between adjacent convex portions 412 .
  • the convex portion 412 does not form a closed annular shape when viewed in the vertical direction (thickness direction of the top plate portion 41).
  • the container bottom portion 12 of the container body 1 is connected to a skirt portion 121 projecting downward from the outer peripheral edge thereof.
  • the outer diameter dimension of the outer peripheral edge of the concentric arcs formed by the plurality of convex portions 412 is slightly smaller than the inner diameter dimension of the skirt portion 121 . According to such a configuration, the convex portion 412 of the cell culture vessel A1 can be fitted into the skirt portion 121 of the other cell culture vessel A1 so that the other cell culture vessel A1 can be stacked. Therefore, a plurality of cell culture vessels A1 can be stacked in a stable posture.
  • the outer lid 4 is made of, for example, an opaque, translucent or transparent plastic material.
  • plastic material include, but are not limited to, polyethylene, polypropylene, polystyrene, methylpentene, and polycarbonate.
  • the cell culture vessel A1 is used for perfusion culture in which the culture solution is automatically supplied at a constant low speed and the same amount of culture solution is discharged at the same time. It should be noted that the cultured cells and the culture solution accommodated in the cell culture container A1 (container body 1) are not particularly limited.
  • each port 24 and the connector 7 are assembled in advance (see Fig. 7).
  • the connector 7 is press-fitted to each of the pair of ports 24 .
  • a perfusion pump (not shown) is connected to one port 24 via a connector 7 and a tube 8
  • a drainage container (not shown) is connected to the other port 24 via a connector 7 and a tube 8.
  • Each tube 8 is passed through the inner side of the outer lid 4 in advance.
  • cells to be cultured are seeded in the container body 1, and a predetermined amount of culture solution is placed in the container body 1.
  • FIG. 8 shows a state in which the container body 1 is filled with the culture medium C.
  • the culture solution C fills the inner space of the container body 1 (the space surrounded by the container bottom portion 12 and the first cylindrical portion 11 of the container body 1 and the first bottom portion 23 of the plug member 2).
  • a small amount of air hereinafter referred to as “bubble B” as appropriate may remain in the inner space of the container body 1 .
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is a flat surface.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 .
  • the air bubbles B in the container body 1 travel along the ceiling surface 231 and near the port 24. move to By operating the perfusion pump in this state, the air bubbles B remaining in the container body 1 can be further moved into the channel 242 through the ceiling recess 232 and discharged to the outside of the cell culture container A1.
  • a new culture solution C is supplied to the container body 1 (cell culture container A1) through one port 24, and the container body 1 (inside the cell culture container A1) is supplied through the other port 24. of the culture solution C is discharged to the outside.
  • the culture solution C in the vessel body 1 is continuously replaced little by little.
  • the cell culture vessel A1 has an outer lid 4 in addition to the vessel body 1 and the plug member 2.
  • a female screw 421 is formed in the third tubular portion 42 of the outer lid 4 fitted onto the first tubular portion 11 of the container body 1 .
  • the first flange portion 22 of the plug member 2 is connected to the upper end 114 of the first cylindrical portion 11 and the outer lid 4 .
  • the assembled state is maintained by being sandwiched between the top plate portion 41 of the .
  • the plug member 2 is in the form of a series of films extending from the first flange portion 22 on the outer peripheral side to the central first bottom portion 23 to the thin portion 238 .
  • the contents (cultured cells and culture medium C) housed in the container body 1 are liquid-sealed. Therefore, in the perfusion culture process, the culture solution perfusion work can be performed while maintaining the closed state of the cell culture vessel A1, and the risk of contamination can be avoided. Therefore, according to the cell culture vessel A1, it is possible to keep the culture solution C in good condition and ensure the quality of the cells.
  • the male screw 111 of the container body 1 (first tubular portion 11) and the female screw 221 of the outer lid 4 (third tubular portion 42) correspond to an example of the "locking means" of the present disclosure.
  • the cell culture vessel A1 includes a cover member 3 in addition to the container body 1 and the plug member 2.
  • the plug member 2 is made of a soft material.
  • the cover member 3 is made of a hard material and is in contact with the plug member 2 in an overlapping manner.
  • the cover member 3 includes a second flange portion 31 that covers the first flange portion 22 of the plug member 2 .
  • the second flange portion 31 is interposed between the first flange portion 22 of the plug member 2 and the top plate portion 41 of the outer lid 4 . Therefore, when the female screw 421 of the outer lid 4 is screwed into the male screw 111 of the container body 1 (first cylindrical portion 11), the top plate portion 41 is in sliding contact with the second flange portion 31 (hard material). slidability becomes good.
  • the cover member 3 includes a fourth tubular portion 32 that covers the inner peripheral surface of the second tubular portion 21 . According to such a configuration, the plug member 2 made of a soft material is pressed against the inner peripheral surface of the container body 1 (first tubular portion 11), and the sealing performance of the cell culture container A1 is enhanced.
  • the cover member 3 also includes a second bottom portion 33 that covers the first bottom portion 23 of the plug member 2 . According to such a configuration, in the perfusion culture process, the first bottom portion 23 (thin portion 238) tries to swell upward due to the pressurizing force of the supplied culture medium C, but the second bottom portion 33 causes the first bottom portion 23 to expand. Deformation of the bottom portion 23 (thin portion 238) is suppressed. As a result, the flow of the culture solution C inside the container body 1 becomes smooth, and the visibility inside the container body 1 is excellent, making it suitable for observing the conditions inside the container body 1 .
  • the port 24 provided on the plug member 2 has an extending portion 241 extending upward from the first bottom portion 23 .
  • the cover member 3 includes a fifth cylindrical portion 34, and the fifth cylindrical portion 34 contacts the outer peripheral surface 241a of the extending portion 241 to cover the outer peripheral surface 241a.
  • the first bottom portion 23 of the plug member 2 includes a thin portion 238 having gas permeability. Therefore, the contents of the container body 1 (cell culture container A1) are maintained in a ventilated state with the outside of the container body 1 (cell culture container A1). Therefore, according to the present embodiment, the contents of the container body 1 (cell culture container A1) can be cultured in an aerated state with the outside. Further, if the cell culture vessel A1 is placed in an incubator under a predetermined gas atmosphere, the gas atmosphere in the incubator can be taken into the container body 1 (cell culture vessel A1).
  • a through hole 331 is formed in the second bottom portion 33 of the cover member 3 so as to pass through the second bottom portion 33 in the thickness direction.
  • the through hole 331 overlaps the thin portion 238 of the plug member 2 when viewed in the vertical direction (first direction). According to such a configuration, while suppressing the deformation of the first bottom portion 23 (the thin portion 238 ) as described above, it is possible to maintain the state of ventilation between the inside and the outside of the container body 1 via the thin portion 238 . .
  • a protrusion 245 that protrudes outward from the outer peripheral surface 241 a of the extension 241 is provided at the tip of the port 24 (extension 241 ).
  • the projecting portion 245 is adjacent to the upper side of the fifth tubular portion 34 and can abut on the upper end of the fifth tubular portion 34 . According to such a configuration, even if a downward pressing force acts on the extending portion 241, the extending portion 241 is prevented from being pushed into the inner space of the container body 1 through the fifth cylindrical portion 34. .
  • a downward pressing force acts on the port 24, but the extending portion 241 is prevented from entering the inner space of the container body 1 unreasonably. Damage to cells in 1 is avoided.
  • FIG. 10 shows a cell culture vessel according to a first modified example of the first embodiment.
  • the same or similar elements as those of the cell culture vessel A1 of the above embodiment are denoted by the same reference numerals as those of the above embodiment, and description thereof will be omitted as appropriate.
  • the cell culture vessel A11 of this modified example differs from the cell culture vessel A1 of the above embodiment mainly in the configuration of the ceiling recess 232 .
  • the ceiling concave portion 232 has a conical portion 233 and a peripheral edge portion 234 .
  • the conical portion 233 is connected to the channel 242 .
  • the cone-shaped portion 233 is a cone-shaped portion formed so that the cross-sectional area increases downward from the position where it connects with the flow path 242 .
  • the peripheral edge portion 234 connects to the conical portion 233 and communicates with the outer peripheral edge 237 of the first bottom portion 23 .
  • the peripheral edge portion 234 is inclined downward as it goes radially outward of the first bottom portion 23 . As shown in FIG.
  • the peripheral edge portion 234 has a substantially fan shape extending radially outward when viewed in the thickness direction (first direction) of the first bottom portion 23 .
  • the annular protrusion 211 is not formed on the outer peripheral portion of the second tubular portion 21 , and the outer peripheral surface of the second tubular portion 21 faces the inner peripheral surface of the first tubular portion 11 . are in direct contact.
  • the cell culture vessel A11 of this modified example is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is flat.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 . According to such a configuration, even if air bubbles remain in the inner space when the inner space of the container body 1 is filled with the culture medium (including cells) prior to perfusion culture, the port 24 is positioned at the upper position.
  • the ceiling recessed portion 232 (peripheral edge portion 234 ) communicates with the outer peripheral edge 237 of the first bottom portion 23 .
  • the cell culture vessel A11 of this modified example has the same effects as the cell culture vessel A1 described above.
  • FIG. 12 is a bottom view showing another example of the plug member 2.
  • FIG. The plug member 2 shown in FIG. 12 differs from the plug member 2 shown in FIG. 11 in the shape of the peripheral portion 234 .
  • the peripheral edge portion 234 is formed to extend along the circumferential direction on the outer peripheral portion of the first bottom portion 23 .
  • the peripheral edge portion 234 is inclined so as to be positioned downward as it goes radially outward of the first bottom portion 23 .
  • the air bubbles can be guided to the flow path 242 along the peripheral edge portion 234 . Thereby, it is possible to more accurately discharge the air bubbles remaining in the inner space of the container body 1 .
  • FIG. 13 shows a cell culture vessel according to a second modification of the first embodiment.
  • the configurations of the first bottom portion 23 of the plug member 2 and the second bottom portion 33 of the cover member 3 are different from those of the cell culture vessel A1 of the above embodiment.
  • the first bottom portion 23 (mainly the thin-walled portion 238) has a curved plate shape, and curves downward (on the other side in the first direction) as it extends radially inward.
  • the ceiling surface 231 is a curved surface that curves downward as it extends radially inward.
  • the second bottom portion 33 is also curved so as to be positioned downward (on the other side in the first direction) as it goes radially inward.
  • the portion of the second bottom portion 33 that overlaps the thin portion 238 of the plug member 2 when viewed in the vertical direction (first direction) is flat (see FIG. 4).
  • the second bottom portion 33 has a curved portion overlapping the thin portion 238 of the plug member 2 when viewed in the vertical direction (first direction).
  • the lower surface of the second bottom portion 33 is in contact with the thin portion 238 .
  • the thin portion 238 may have a curved shape in its natural state, but is not limited to this.
  • the thin portion 238 may be flat in its natural state, and may be curved when covered with the second bottom portion 33 of the cover member 3 .
  • the cell culture container A12 of this modified example is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is curved.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 . According to such a configuration, even if air bubbles remain in the inner space when the inner space of the container body 1 is filled with the culture medium (including cells) prior to perfusion culture, the air bubbles remain on the curved ceiling surface 231.
  • the cell culture vessel A12 of this modified example has the same effects as the cell culture vessel A1 described above.
  • FIG. 14 shows a cell culture vessel according to a third modified example of the first embodiment.
  • the configuration of one of the pair of ports 24 is different from that of the cell culture vessel A1 of the above embodiment.
  • the port 24 located on the left side of FIG. 14 is the same as that of the above embodiment, but the configuration of the port 24 located on the right side of the figure is different.
  • the port 24 located on the right side of FIG. 14 has a protrusion 247 extending below the first bottom 23 .
  • the ceiling recess 232 is not formed around the projecting portion 247 in the first bottom portion 23 .
  • the cell culture vessel A13 of this modified example is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is flat.
  • the first bottom portion 23 is formed with a ceiling recess 232 recessed upward from the ceiling surface 231 , and the ceiling recess 232 is connected to a flow path 242 of one port 24 (left side in FIG. 14).
  • one port 24 (left side in FIG. 14) ) is positioned at the top, air bubbles in the container body 1 move along the ceiling surface 231 to the vicinity of the one port 24 . Then, the air bubbles float in the channel 242 through the ceiling recess 232 . Thereby, the air bubbles remaining in the container body 1 can be discharged to the outside of the cell culture container A13. Then, when perfusion culture is performed, a new culture solution C is supplied to the container body 1 (cell culture container A13) through the port 24 located on the right side of FIG. The culture solution inside the body 1 (inside the cell culture container A13) is discharged to the outside. In the perfusion culture process, the culture solution in the container body 1 is continuously replaced little by little.
  • the cell culture vessel A13 of this modified example has the same effects as the cell culture vessel A1 described above.
  • FIG. 15 shows a cell culture vessel according to a fourth modified example of the first embodiment.
  • the configuration of the second bottom portion 33 of the cover member 3 is different from that of the cell culture vessel A1 of the above embodiment.
  • one through hole 331 is formed in the second bottom portion 33 .
  • the diameter of the through hole 331 is remarkably increased as compared with the above embodiment.
  • the through hole 331 overlaps most of the thin portion 238 of the plug member 2 when viewed in the vertical direction (first direction).
  • the cell culture vessel A14 of this modification is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is flat.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 . According to such a configuration, even if air bubbles remain in the inner space when the inner space of the container body 1 is filled with the culture medium (including cells) prior to perfusion culture, the port 24 is positioned at the upper position.
  • the cell culture vessel A14 of this modified example has the same effects as the cell culture vessel A1 described above.
  • FIG. 16 shows a cell culture vessel according to a second embodiment of the present disclosure.
  • the cell culture vessel A2 of this embodiment differs from the cell culture vessel A1 of the above embodiment in the configuration of the port 24 .
  • the port 24 has a first channel 243, a second channel 244, and a non-penetrating portion 246 instead of the channel 242 of the above embodiment.
  • the flow path 242 in the first embodiment is a communication path that communicates with the outside and the inner space of the container body 1, but in the present embodiment, the first flow path 243 and the second flow path 244 are , are separated from each other in the vertical direction (first direction).
  • the first channel 243 is located near the upper portion of the port 24 and is open to the outside.
  • the second channel 244 is located near the bottom of the port 24 , opens to the inner space of the container body 1 , and is connected to the ceiling recess 232 .
  • the first flow path 243 and the second flow path 244 overlap each other when viewed in the vertical direction (first direction).
  • the non-penetrating portion 246 is a portion interposed between the first channel 243 and the second channel 244 .
  • the cell culture vessel A2 of the present embodiment is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of the culture solution is discharged at the same time.
  • the introduction of cells and culture solution into the inner space of the container body 1 and the discharge of the waste liquid are performed, for example, by attaching the plug member 2, the cover member 3 and the outer lid 4 to the container body 1, and After A1 is brought into an assembled state, the non-penetration portion 246 of the port 24 is pierced with an injection needle (not shown).
  • the tip of the injection needle is arranged in the second flow path 244 immediately below the non-penetrating portion 246 .
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is a flat surface.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 .
  • the port 24 provided on the plug member 2 has an extending portion 241 extending upward from the first bottom portion 23 .
  • the cover member 3 includes a fifth cylindrical portion 34, and the fifth cylindrical portion 34 contacts the outer peripheral surface 241a of the extending portion 241 to cover the outer peripheral surface 241a.
  • the cell culture vessel A2 of this embodiment has the same effects as the cell culture vessel A1 described above.
  • FIG. 17 shows a cell culture vessel according to the third embodiment of the present disclosure.
  • the cell culture vessel A3 of this embodiment does not include the cover member 3 unlike the cell culture vessel A1 of the first embodiment. That is, the cell culture vessel A3 has a vessel body 1, a plug member 2 and an outer lid 4. As shown in FIG.
  • the configurations of the vessel body 1 and the outer lid 4 are the same as in the first embodiment.
  • the configuration of the plug member 2 is significantly different from that of the first embodiment.
  • the plug member 2 is configured with a first portion 2A and a second portion 2B.
  • the second part 2B is made of a soft material having flexibility and elasticity, and has a second tubular part 21 and a first flange part 22 .
  • the second portion 2B is interposed between the first portion 2A and the first cylindrical portion 11 of the container body 1.
  • a pair of annular projections 211 are formed on the outer peripheral portion of the second cylindrical portion 21 with a gap therebetween in the vertical direction (first direction).
  • the first portion 2A includes a first bottom portion 23, a port 24, an inner cylindrical portion 26 and an upper flange portion 27.
  • the first bottom portion 23 has a substantially constant thickness. Accordingly, in this embodiment, unlike the first embodiment, the first bottom portion 23 does not have the thin portion 238 .
  • a ceiling recess 232 formed in the first bottom portion 23 has a conical portion 233 and a peripheral portion 234 .
  • the conical portion 233 is connected to the channel 242 .
  • the cone-shaped portion 233 is a cone-shaped portion formed so that the cross-sectional area increases downward from the position where it connects with the flow path 242 .
  • the peripheral edge portion 234 is connected to the conical portion 233 and communicates with the outer peripheral edge of the first bottom portion 23 .
  • the peripheral edge portion 234 is inclined downward as it goes radially outward of the first bottom portion 23 .
  • the inner tubular portion 26 extends upward from the outer peripheral edge of the first bottom portion 23 and has a substantially cylindrical shape.
  • the inner tubular portion 26 covers the inner peripheral surface of the second tubular portion 21 .
  • the upper flange portion 27 has a substantially annular shape and extends radially outward from the upper end of the inner cylindrical portion 26 .
  • the upper flange portion 27 overlaps the first flange portion 22 and the first tubular portion 11 when viewed in the thickness direction of the upper flange portion 27 .
  • the first part 2A is made of a hard material, such as a translucent or transparent plastic material.
  • a hard material such as a translucent or transparent plastic material.
  • the plastic material include, but are not limited to, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other transparent materials.
  • the cell culture vessel A3 of the present embodiment is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of the culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is flat.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 . According to such a configuration, even if air bubbles remain in the inner space when the inner space of the container body 1 is filled with the culture medium (including cells) prior to perfusion culture, the port 24 is positioned at the upper position.
  • the ceiling recessed portion 232 (peripheral edge portion 234) communicates with the outer peripheral edge of the first bottom portion 23.
  • the air bubbles are removed from the outer peripheral edge of the first bottom portion 23 or the outer peripheral edge of the second cylindrical portion 21. Even if it reaches the edge side, the air bubble can be guided to the channel 242 along the peripheral edge portion 234 . Thereby, it is possible to more accurately discharge the air bubbles remaining in the inner space of the container body 1 .
  • the cell culture vessel A3 of the present embodiment has the same structure as the cell culture vessel A1 of the first embodiment described with reference to FIGS. It has the same function and effect as.
  • FIG. 18 shows a cell culture vessel according to a fourth embodiment of the present disclosure.
  • the cell culture vessel A4 of this embodiment does not have the cover member 3 and the outer lid 4.
  • the cell culture vessel A4 has a container body 1 and a plug member 2.
  • FIG. 18 shows a container body 1 and a plug member 2.
  • the external thread 111 is not formed on the outer peripheral surface of the first cylindrical portion 11 of the container body 1 .
  • the plug member 2 is, for example, a molded rubber product, and includes a second cylindrical portion 21, a first flange portion 22, a first bottom portion 23 and a pair of ports 24.
  • the plug member 2 is made of a soft material having flexibility and elasticity.
  • the material constituting the plug member 2 is the same as that of the first embodiment.
  • the annular protrusion 211 is not formed on the outer peripheral portion of the second tubular portion 21 .
  • the outer diameter dimension of the second tubular portion 21 is larger than the inner diameter dimension of the first tubular portion 11 in the natural state.
  • the second tubular portion 21 configured as described above is press-fitted into the first tubular portion 11 .
  • the outer peripheral surface of the second cylindrical portion 21 is in close contact with (abuts) the inner peripheral surface of the first cylindrical portion 11 to form a tight seal.
  • the first bottom portion 23 does not have the thin portion 238 .
  • the ceiling concave portion 232 formed in the first bottom portion 23 has a conical shape and is formed across the port 24 (extending portion 241) from the first bottom portion 23. As shown in FIG.
  • the cell culture vessel A4 of this embodiment is used for perfusion culture in which cultured cells and a culture solution are accommodated, the culture solution is automatically supplied at a constant low speed, and the same amount of the culture solution is discharged at the same time.
  • the ceiling surface 231 of the first bottom portion 23 of the plug member 2 is flat.
  • a ceiling recess 232 that is recessed upward from the ceiling surface 231 is formed in the first bottom portion 23 , and the ceiling recess 232 is connected to the flow path 242 of the port 24 . According to such a configuration, even if air bubbles remain in the inner space when the inner space of the container body 1 is filled with the culture medium (including cells) prior to perfusion culture, the port 24 is positioned at the upper position.
  • the cell culture vessel A4 of the present embodiment has the same structure as the cell culture vessel A1 of the first embodiment described with reference to FIGS. It has the same function and effect as.
  • the cell culture vessel according to the present disclosure is not limited to the above embodiments.
  • the specific configuration of each part of the cell culture vessel of the present disclosure can be changed in various ways.
  • A1, A11, A12, A13, A14, A2, A3, A4 cell culture vessel
  • B air bubble
  • C culture solution
  • 1 container body
  • 11 first cylindrical portion
  • 110 opening
  • 111 male screw (locking means)
  • 112 slit
  • 113 extension piece
  • 114 upper end (first direction one side end of the first cylindrical portion)
  • 12 container bottom
  • 121 skirt portion
  • 2 plug member
  • 2A first part
  • 2B second part
  • 21 second tubular part
  • 232 ceiling concave part
  • 233 cone shape portion 234: peripheral portion 235: outer peripheral side thick portion 237: outer peripheral edge 238: thin portion
  • 241: extension portion 241a outer peripheral surface
  • 242 flow path 243: first Flow path 244: Second flow path 245: Protruding portion 246: Non-penetrating portion 247: Protruding portion 26: Inner cylindrical

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Abstract

La présente invention concerne un récipient de culture cellulaire adapté pour améliorer les difficultés lors de la réalisation d'une culture par perfusion dans un état fermé. Le récipient de culture cellulaire est équipé d'un corps de récipient (1) ayant une ouverture (110) sur l'extrémité supérieure et une première partie cylindrique (11) s'étendant verticalement, et d'un élément formant bouchon (2) capable de sceller l'ouverture (110). L'élément formant bouchon (2) comprend les éléments suivants : une seconde partie cylindrique (21) venant en butée contre la surface périphérique intérieure et fermant la première partie cylindrique (11) ; une première partie formant bride (22) s'étendant radialement vers l'extérieur depuis l'extrémité supérieure de la seconde partie cylindrique (21) et bloquant l'extrémité supérieure (114) de la première partie cylindrique (11) ; et une première partie formant fond (23) s'étendant radialement vers l'intérieur depuis l'extrémité inférieure de la seconde partie cylindrique (21) et bloquant l'intérieur de la seconde partie cylindrique (21) lorsqu'elle est observée dans la direction verticale. La première partie inférieure (23) est pourvue d'une pluralité d'orifices (24) ayant chacun un chemin d'écoulement (242) menant à l'extérieur et à l'espace intérieur du corps de récipient (1). La première partie inférieure (23) a une surface de plafond (231) orientée vers le bas, et des évidements de plafond (232) en retrait vers le haut de la surface de plafond (231) sont formés dans la première partie inférieure (23). Les évidements du plafond (232) se raccordent aux voies d'écoulement (242).
PCT/JP2021/009546 2021-03-10 2021-03-10 Récipient de culture cellulaire Ceased WO2022190261A1 (fr)

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PCT/JP2021/009546 WO2022190261A1 (fr) 2021-03-10 2021-03-10 Récipient de culture cellulaire
JP2023504964A JP7765842B2 (ja) 2021-03-10 2021-03-10 細胞培養容器

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013543738A (ja) * 2010-11-22 2013-12-09 コーニング インコーポレイテッド 細胞培養装置のための密閉アセンブリ
WO2019082261A1 (fr) * 2017-10-24 2019-05-02 株式会社サンプラテック Conteneur de cellules
WO2021014642A1 (fr) * 2019-07-25 2021-01-28 株式会社サンプラテック Récipient destiné à recevoir une solution de culture

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109219440A (zh) 2016-04-04 2019-01-15 贝塔O2技术有限公司 用于植入具有抗炎和血管化能力的细胞的可植入设备及其制造方法
JP7319976B2 (ja) 2018-07-10 2023-08-02 テルモ株式会社 検体回収機構を具備する移植片収容デバイス

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013543738A (ja) * 2010-11-22 2013-12-09 コーニング インコーポレイテッド 細胞培養装置のための密閉アセンブリ
WO2019082261A1 (fr) * 2017-10-24 2019-05-02 株式会社サンプラテック Conteneur de cellules
WO2021014642A1 (fr) * 2019-07-25 2021-01-28 株式会社サンプラテック Récipient destiné à recevoir une solution de culture

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