WO2022176980A1 - Procédé de production cellulaire - Google Patents

Procédé de production cellulaire Download PDF

Info

Publication number
WO2022176980A1
WO2022176980A1 PCT/JP2022/006663 JP2022006663W WO2022176980A1 WO 2022176980 A1 WO2022176980 A1 WO 2022176980A1 JP 2022006663 W JP2022006663 W JP 2022006663W WO 2022176980 A1 WO2022176980 A1 WO 2022176980A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
container
cell
culture
square
Prior art date
Application number
PCT/JP2022/006663
Other languages
English (en)
Japanese (ja)
Inventor
一成 南
和磨 庄司
Original Assignee
株式会社マイオリッジ
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 株式会社マイオリッジ filed Critical 株式会社マイオリッジ
Publication of WO2022176980A1 publication Critical patent/WO2022176980A1/fr

Links

Images

Classifications

    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to, for example, cell production methods, culture vessels, culture apparatuses, enzyme treatment methods, and the like.
  • FIG. 1 of Patent Document 1 describes a cell culture apparatus equipped with stirring blades for stirring a culture solution.
  • FIG. 1 of Patent Document 2 describes a double circular dish having an annular flow path, and Example 1 describes that cells were cultured by spinning using the dish.
  • Patent Document 1 uses stirring blades for culture, cell damage is likely to occur due to physical collision between the cells and the stirring blades.
  • Patent Document 2 since the container has a ring shape, there is a dead space in the center. Therefore, the culture capacity was limited.
  • the purpose of the present invention is, for example, to provide an excellent method for producing cells.
  • a cell production method comprising the step of horizontally rotating a container containing cells and a culture medium, the container having a square bottom shape. According to this production method, excellent cell culture can be performed.
  • a cell culture device comprising a medium containing section and a rotating section for horizontally rotating the medium containing section, wherein the medium containing section has a square bottom shape.
  • a cell culture device is provided. Excellent cell culture can be performed using this device.
  • a horizontal rotating cell culture vessel comprising a bottom surface, a top surface and a side surface, the bottom surface having a square shape and the top surface having 1 to 4 openings.
  • a rotating cell culture vessel is provided. Excellent cell culture can be performed using this vessel.
  • a method for enzymatic treatment of cells which includes the step of horizontally rotating a container containing cells and an enzyme solution, and the bottom shape of the container is square. According to this treatment method, an excellent enzymatic treatment can be performed.
  • an apparatus for treating cells with enzymes which includes a medium container and a rotation unit that horizontally rotates the medium container, and the medium container has a square bottom surface.
  • a cellular enzymatic treatment device is provided. Excellent enzymatic treatments can be performed with this device.
  • a horizontal rotating cell enzymatic treatment vessel comprising a bottom surface, a top surface and a side surface, the bottom surface having a square shape and the top surface having 1 to 4 openings, A horizontal rotating cell enzymatic treatment vessel is provided. Excellent cell culture can be performed using this vessel.
  • FIG. 1 is a bar graph showing the proliferation rate of iPS cells per unit area in each vessel and swirling conditions shown in Examples.
  • FIG. 2 is a bar graph showing the total iPS cell yield in each container and swirling conditions shown in Examples.
  • FIG. 3 is a bar graph showing the results of comparison of cardiomyocyte differentiation potential of iPS cells cultured with 50 rpm rotation shown in Examples.
  • FIG. 4 is a photograph of beads with adhered mesenchymal stem cells shown in Examples.
  • FIG. 5 is a diagram showing the distribution of beads to which mesenchymal stem cells are adhered when a 15-cm-diameter circular container shown in Examples is subjected to swirling culture.
  • FIG. 1 is a bar graph showing the proliferation rate of iPS cells per unit area in each vessel and swirling conditions shown in Examples.
  • FIG. 2 is a bar graph showing the total iPS cell yield in each container and swirling conditions shown in Examples.
  • FIG. 3 is a bar graph showing the results of comparison of cardio
  • FIG. 6 is a diagram showing the distribution of beads to which mesenchymal stem cells are adhered when a square container with a side of 22 cm is subjected to swirling culture as shown in the example.
  • FIG. 7 is a photograph of floating iPS cell clusters produced by swirl culture in a circular container with a diameter of 15 cm shown in the example.
  • FIG. 8 is a photograph of floating iPS cell clusters produced by swirling the 22 cm square container shown in the example.
  • FIG. 9 is a bar graph showing the proliferation rate of mesenchymal stem cells per unit area in each vessel shown in Examples and under 50 rpm turning conditions.
  • FIG. 10 is a bar graph showing the total mesenchymal stem cell yield in each vessel shown in Examples and under the condition of 50 rpm.
  • FIG. 7 is a photograph of floating iPS cell clusters produced by swirl culture in a circular container with a diameter of 15 cm shown in the example.
  • FIG. 8 is a photograph of floating iPS cell clusters produced by swirl
  • FIG. 11 is a bar graph showing the survival rate of mesenchymal stem cells in each container condition when performing the swirling enzyme treatment shown in Examples.
  • FIG. 12 is a bar graph showing the number of mesenchymal stem cells per volume of enzyme solution under each container condition when the swirling enzyme treatment shown in the Examples is performed.
  • FIG. 13 is a diagram of the first container of one embodiment.
  • FIG. 14 is a front view etc. of the first container of one embodiment.
  • FIG. 15 is a front view etc. of the first container of the embodiment with the screw cap removed.
  • FIG. 16 is a diagram of four stacked first containers of one embodiment.
  • FIG. 17 is a front view of a state in which four first containers of one embodiment are stacked.
  • FIG. 18 is an image photograph of a state in which four first containers of one embodiment are stacked.
  • FIG. 19 is an illustration of a second container according to one embodiment.
  • FIG. 20 is a diagram of two stacked second containers of one embodiment.
  • One embodiment of the present invention is a novel cell production method.
  • This production method includes, for example, a cell production method including a step of horizontally rotating a container containing cells and a culture medium, and the container having a square bottom shape.
  • a cell production method including a step of horizontally rotating a container containing cells and a culture medium, and the container having a square bottom shape.
  • this production method for example, from the viewpoint of at least one of an improvement in cell growth rate, an improvement in the number of cells after culture, an improvement in cell dispersion, uniformity in cell shape, uniformity in cell size, or reduction in shear stress, Excellent cell culture can be achieved.
  • the cell culture method using the square dish of the example is superior to the cell culture method using the round dish in at least one of these effects.
  • iPS cells and MSCs are vulnerable to shear stress, so it is preferable to rotate at a low speed during culture. I have too many problems.
  • the cells can be kept in a state in which it is difficult for the cells to gather at the center (that is, they are dispersed) even when the cells are rotated at a low speed, which causes less shear stress on the cells.
  • it was also found that, in the case of low-speed rotation, it is preferable to lower the liquid level of the culture medium, and furthermore, it is preferable to set the height of the container low from the viewpoint of large-scale culture.
  • the method for producing cells includes, for example, supplying a medium to a container, adjusting a cell suspension containing cells and a medium, supplying a cell suspension to a container, It may include a step of sealing, a step of placing the container on a rotating device, a step of rotating the container, a step of rotating and rocking culturing the cells, a step of suspension culturing the cells, or a step of collecting the cells from the container.
  • the method for producing cells includes, for example, a step of replacing the medium in the container with an enzyme (e.g., proteolytic enzyme) solution, a step of enzymatically treating the cells, a step of dispersing the cells, a step of A step of converting clumps to single cells or rotating a container containing a cell suspension containing enzymatically treated or dispersed cells may be included.
  • Collecting the cells from the container may comprise, for example, pipetting up the medium or the cells.
  • the rotating device may, for example, be a horizontal swivel device.
  • the method for producing cells includes the step of horizontally rotating a container having a square bottom shape, so that the medium may rebound in waves during cell culture.
  • the wave bounce is then caused by the movement of the medium between the two opposing sides, causing the waves to bounce back and forth between the two sides.
  • a container having a circular bottom shape such rebounding of waves does not occur.
  • Such wave bouncing may be used as an indicator of an environment in which cells tend to disperse during culture.
  • cells may be, for example, floating cells, cell clusters (spheroids), or single cells.
  • the production method described above is particularly useful for culturing spheroids that easily fuse because cells can easily maintain a dispersed state.
  • the production method described above is particularly useful in culturing iPS cells and MSCs because of its low shear stress.
  • the cells may also be mammalian cells. Mammals include, for example, humans, monkeys, rodents (mice, hamsters, etc.), and the like.
  • Cells include, for example, stem cells or somatic cells. Stem cells include, for example, cells that have the ability to self-renew and differentiate into other cell types.
  • Stem cells include pluripotent stem cells, multipotent stem cells, and unipotent stem cells.
  • Pluripotent stem cells include, for example, iPS cells or ES cells.
  • the pluripotent stem cells may express any undifferentiated marker at the same level or higher than, for example, human induced pluripotent stem cell line 253G1 (HPS0002).
  • Multipotent stem cells include, for example, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, neural stem cells, and the like.
  • Unipotent stem cells include, for example, muscle stem cells, melanocyte stem cells, and the like.
  • Somatic cells include, for example, cells derived from skin, heart, liver, lung, stomach, intestine, kidney, uterus, brain, blood, or mesenchymal tissue.
  • Cells also include, for example, T cells and CHO cells.
  • Cells may be attached to a carrier or material (eg, plastic material (eg, plastic beads)).
  • the medium in one embodiment of the present invention may be a liquid medium.
  • the medium may be, for example, a medium for stem cells (eg, Essential 8 Medium), a medium for mammalian cells (eg, GIBCO Advanced medium (Thermo Fisher)), or the like.
  • examples of media include serum media (e.g., 10% FBS), balanced salt solutions (e.g., PBS, etc.), basal media (MEM, etc.), complex media (e.g., RPMI 1640, etc.), serum-free media, etc. may
  • the vessel comprises a cell culture vessel.
  • the container type may be, for example, a dish type, petri dish type, or flask type.
  • a container may have a bottom surface, sides, and a top surface.
  • the shape of the bottom, side, or top of the container may be square.
  • a square shape can be distinguished from a circular shape in that it has four sides.
  • the dish with product number MS-12450 (Sumitomo Bakelite) (22.4 cm on each side (inner dimensions), 500 cm 2 in area, 2.4 cm in height (inner dimensions), 0.4 cm liquid level for 200 ml of culture medium) has a square bottom. It is a type of mold-shaped container.
  • Square-shaped shapes include, for example, square-shaped or rectangular-shaped shapes.
  • Square-type shapes include shapes where the four sides are substantially the same length. Rectangular-type shapes include shapes that have two sets of two sides of substantially the same length (but not square-type shapes).
  • the shape of the bottom surface is particularly preferably a square shape from the viewpoint of improving the cell proliferation rate, improving the number of cells after culture, or improving cell dispersion.
  • the number of bottom and top surfaces may be one each, and the number of side surfaces may be four.
  • the bottom surface may be the lowest surface that is in constant contact with the medium during culture.
  • the side surface may form a surface rising upward with respect to the bottom surface. Alternatively, the side surface may be positioned substantially perpendicular to the bottom surface.
  • a part of the side surface may be in contact with the medium during culture, and the remaining part of the surface may not be in contact with the medium.
  • the side surfaces may contact the bottom surface at the bottom end and the top surface at the top end.
  • the sides may have two sets of sides in parallel relationship.
  • the top surface may lie parallel to the bottom surface.
  • the top surface may be the uppermost surface that is not in constant contact with the medium during culture.
  • the top surface may be positioned substantially perpendicular to the side surface.
  • the container may have a closed space inside.
  • the bottom, sides, and top may comprise flat surfaces.
  • the face may consist of flat walls.
  • the quadrangular shape may have two sets of parallel sides.
  • any side may be called vertical, and a side that is in contact with the vertical (or a side that is not parallel to the vertical) may be called horizontal.
  • the square shape is a rectangular shape, the shortest side may be referred to as length and the longest side as width.
  • the ratio of the vertical and horizontal lengths of the square shape is, from the viewpoint of improving the cell growth rate, improving the number of cells after culture, or improving cell dispersion, when the vertical is 1, the horizontal is 1 ⁇ 1.05 is preferred, and 1 to 1.01 is particularly preferred.
  • the ratio of the length to the width of a rectangular shape is 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.1, 1.2, 1.3, 1.4, or 1.5 when the length is 1. or within any two of those values.
  • the length of one side of the square shape is particularly preferably 20 to 40 cm from the viewpoint of improving the cell growth rate, improving the number of cells after culturing, or improving cell dispersion.
  • the length of one side of the square shape is, for example, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 cm. and may be within any two of those values.
  • one side of the square shape of the bottom is large, for example, 20 cm or more. Edges include straight lines.
  • the length of the quadrangular shape may be positioned substantially perpendicular to the width. Adjacent sides of the four sides of the quadrangular shape may be positioned substantially perpendicular to each other.
  • substantially right angles include substantially right angles.
  • the angle at which the sides to the bottom of the container, the sides to the top, and the sides to the length of the bottom shape lie, or approximately right angles, are, for example, 90 degrees plus or minus 15, 10, 5, 3, 2, 1, 0.5, or 0 degrees.
  • the shape of the quadrangle may be one that can be recognized as a quadrangle as a whole. may be connected.
  • the total length of the four sides of the square shape may be 40, 50, 75, 100, 150, 200, 300, or 500 times the total length of the joints, any value thereof greater than or equal to or within any two of those values.
  • the total length of the straight portions of the square shape may be 40, 50, 75, 100, 150, 200, 300, or 500 times the total length of the non-linear portions, or greater than any of those values, or It may be within any two of those values.
  • the rotation may be horizontal rotation.
  • Rotation includes turning.
  • a horizontal plane may include a plane perpendicular to the direction in which the earth's gravitational force acts.
  • Horizontal rotation when used for culturing includes a mode of rotating on a plane that is substantially vertical or nearly vertical to the direction in which gravity acts.
  • the substantially vertical or substantially vertical plane may include a slight inclination due to the design of the rotating device or due to slight inclination of the table on which the rotating device is placed. This tilt may be, for example, 3, 2, 1, 0.5, 0.2, or 0.1 degrees above or below the vertical plane.
  • the position of the rotational axis of rotation of the rotator may not coincide with the rotational axis of rotation of the container.
  • the location of the axis of rotation or center of rotation of the container may be within the container.
  • the rotational speed is preferably low, such as 60 rpm or less, from the viewpoint of improving the cell growth rate, improving the number of cells after culture, or improving cell dispersion.
  • 60 rpm or less is preferably 30 to 60 rpm, particularly preferably 40 to 55 pm.
  • Rotational speeds are, for example, or 200 rpm, or greater than any of those values, or within any two of those values.
  • the rotation frequency is preferably 0.5 to 1.0 Hz, particularly preferably 0.666 to 0.917 Hz, from the viewpoint of improving the cell growth rate, improving the number of cells after culture, or improving cell dispersion. .
  • the frequency may be, for example, 0.02, 0.15, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.3, 1.5, 1.6, 1.67, 2, 2.33, 2.67, 3, or 3.33 Hz; It may be greater than or equal to either of those values, or within a range of any two of those values.
  • the turning diameter (rotational amplitude) of rotation is preferably 1 to 5 cm, preferably 2 to 3 cm, from the viewpoint of improving the cell growth rate, improving the number of cells after culture, or improving cell dispersion. is particularly preferred.
  • the radius of gyration may be, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 30, 40, or 50 cm. May be within any two values.
  • the liquid level of the medium in the container is low in order to prevent deterioration of the oxygen environment due to sinking of the cells.
  • the liquid level of the medium is low, the ratio of the contact area between the medium and oxygen to the medium volume is large, and deterioration of oxygen supply can be prevented.
  • the height of the liquid surface of the medium in the container is preferably 3 cm or less, more preferably 1.5 cm or less, and particularly preferably 1.2 cm or less.
  • the liquid level is, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.5, 2, 3, 4, 5, 10, or 20 cm, less than or within any two of those values.
  • the ratio of the liquid level of the culture medium in the container to the height of the container is preferably 0.5 or less, particularly preferably 0.4 or less. This ratio may be, for example, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5, less than or within any two of these values.
  • the ratio of the height of the liquid surface of the medium in the container to the length of one side (e.g., vertical or horizontal) of the bottom shape of the container is preferably 0.2 or less, and 0.06.
  • This ratio may be, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.1, 0.15, 0.2, 0.25, or 0.3, less than or equal to any of those values, or less than or equal to any two of those values. may be within the range.
  • the height of the container When setting the liquid level of the medium in the container low, the height of the container can also be set low accordingly. In addition, by setting the height of the container low, the stability when stacking a plurality of containers is improved. In addition, by setting the height of the container low, a larger number of containers can be installed in the culture facility (for example, an incubator), enabling mass culture. From the viewpoint of stability when the containers are stacked, the height of the containers is preferably equal to or less than the length of one side of the bottom shape. In addition, from the viewpoint of providing a medium for mass culture, improving the cell growth rate, improving the number of cells after culture, or improving cell dispersion, etc.
  • the thickness ratio is preferably 0.07 to 0.35, particularly preferably 0.08 to 0.3.
  • This ratio may be, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15, 0.18, 0.2, 0.25, 0.26, 0.3, 0.35, 0.4, 0.5, 0.6 or 1 in height, It may be less than or equal to either of those values, or within a range of any two of those values.
  • one side of the bottom shape includes a long side (longest side) or a short side (shortest side), preferably the short side.
  • the container height may be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 80, or 100 cm. , may be within the range of any two of them.
  • the height of the container may, for example, be expressed as the shortest distance between the bottom and top of the sides.
  • the lowermost portion may be the position where the side surfaces and the bottom surface are in contact
  • the top portion may be the position where the side surface and the upper surface are in contact.
  • the height may be represented by the shortest distance between the bottom surface and the top surface.
  • the height may be represented by the length from the top to the bottom of the container when viewed from the side. Height may be expressed as the shortest distance between the bottom surface and the bottom edge of the cap if there is a cap on the top surface.
  • the volume of the medium in the container is, for example, 4, 5, 8, 10, 20, 50, 60, 100, 200, 300, 400, 600, 800, 1000, 1500, or 2000 mL. or more than any of these values, or within the range of any two of these values.
  • the volume of the medium in the container is, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 1.5, 2, 4, 6, 8, or 10 mL per 1 cm 2 of bottom area. It may be less than any of these values, or within any two of those values.
  • the fill factor of the medium in the container may be, for example, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, any value below, or The liquid volume may be within the range of any two of these values.
  • the fill factor can be expressed as the ratio of medium volume to container volume.
  • the cell culture may be performed, for example, for 1, 5, 10, 20, 24, 48, 72, 96, 120, or 150 hours, any of these values or less, or any of them. A range of two values may be implemented.
  • Cell culture may be performed, for example, in a cell culture incubator (eg, 37° C., 5% CO 2 ). Alternatively, culture conditions used in conventional cell culture can be appropriately selected.
  • the cell concentration in the medium is, for example, 5 x 107 , 10 x 107 , 30 x 107 , 50 x 107 , 80 x 107 , 100 x 107 , or 150 x 10 It may be 7 /L, or it may be any value or more, or the liquid amount may be within the range of any two of these values.
  • the culture time may be, for example, 0.25, 0.5, 1, 5, 12, 24, 48, 72, 96, 168, or 240 hours, or May be within any two values.
  • the container may have an opening.
  • the opening may be in the top, side, or bottom of the container.
  • a biological sample (such as cells) or a culture medium can be injected from the space outside the container into the space inside the container through the opening.
  • the opening may have a cylindrical structure or a circular structure in horizontal cross-section.
  • the opening may have a port or a cap.
  • the port may have a structure (for example, a cylindrical structure) that communicates the space inside the container with the space outside the container.
  • the cap may have a structure that blocks the space inside the container from the space outside the container by covering the port.
  • the cap can seal the inside of the container by sealing the opening.
  • the cap may have, for example, a cylindrical structure with one of the upper end surface and the lower end surface closed.
  • the cap may be, for example, a screw cap.
  • the openings may be located, for example, at the four corners of the outer quadrangular shape on the top or sides.
  • the openings may, for example, be located on the diagonals of the outer quadrangular shape on the top or sides. If the opening has a cylindrical structure or a structure with a circular horizontal cross-section, the central axis of the circle may, for example, be located on the diagonal of the outer square shape of the top surface or side surface. .
  • the number of openings is preferably four. In this case, as shown in FIG. 16, which will be described later, it is possible to stably stack a plurality of containers, or the center of gravity is less likely to be biased when rotating and rocking.
  • the number of openings is preferably one or two.
  • the containers can be stacked as they are, and a large culture area can be secured.
  • the number of openings may be, for example, 1, 2, 3, 4, 5, 6, 7, or 8, or any number between any two of these values.
  • the height of the opening may be, for example, 1, 2, 2.5, 3, 3.5, 4, 5, or 10 cm, or within any two of these values. The height of the opening may be measured with the surface side in contact with the opening as the lower end.
  • the opening is a cylindrical structure
  • its diameter may be, for example, 1, 2, 2.5, 3, 3.5, 4, 5, or 10 cm and within any two of these values. good too.
  • the ratio of the diameter of the opening to the length of one side (e.g., vertical or horizontal) of the bottom shape is 0.05, 0.1, 0.11, 0.115, 0.12, 0.125, when one side of the bottom shape is 1. It may be 0.13, 0.135, 0.14, 0.145, or 0.15, less than or within any two of those values.
  • the shape, length, or ratio of the bottom, side, or top surface of the container described above or below is the shape and length of the inside of the container (the side that contacts the space in the container, or the side that accommodates the culture medium in the container). or may be applied to ratios. For example, a statement that the bottom surface of the container has a square shape includes that the inner bottom surface of the container has a square shape.
  • the area of the bottom of the container may be, for example, 1, 4, 9, 25, 50, 100, 500, 1000, 2500, 5000, or 10000 cm 2 , any two of which It can be any number within a range of values.
  • the bottom and side surfaces of the container may be positioned at substantially right angles.
  • the bottom and side surfaces may be directly connected to form an angle, or may be connected via a connection (eg, linear or arc-shaped).
  • the connecting part is the part between the straight part of the bottom surface and the straight part of the side surface when the container is viewed from the side, which is not on the extension of the straight part of the bottom but on the extension of the straight part of the bottom.
  • a lateral side includes any lateral side or four lateral sides.
  • the curve radius R is preferably small from the viewpoint of improving cell dispersion, for example, preferably 0.5 cm or less, and particularly preferably 0.3 cm or less.
  • the curve radius may be, for example, 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 cm, below any of those values, or within any two of those values. There may be.
  • the curve radius may be the curve radius of the curved portion connecting the bottom surface and the side surface when the container is viewed from the side surface.
  • the curve radius is, for example, the curve radius at the position where the curve radius is the smallest within the curve portion.
  • the top and side surfaces may be directly connected to form an angle, or may be connected via a connection (eg, straight or arc-shaped).
  • the side and another side may be directly connected to form an angle, or may be connected via a connection (eg, straight or arc-shaped).
  • the constituent material of the container includes, for example, synthetic resin, natural resin, or glass.
  • Synthetic resins include, for example, polystyrene resins, polypropylene resins, polyethylene resins, and the like.
  • the inner surface of the container may be composed of a cell non-adhesive resin or coated with a cell anti-adhesion agent.
  • the container may be manufactured by molding techniques, 3D printing, or the like.
  • Fig. 13 shows the first container as an example of a container.
  • the container has a square bottom 1 and top 2 shape. Side 3 has a rectangular shape.
  • the top surface 2 is provided with four openings 4. Since the opening 4 is provided on the upper surface 2, the medium is less likely to spill, and the operability of medium replacement and cell collection is excellent.
  • the opening 4 has a port 6 inside, and a screw cap 5 is installed so as to cover it.
  • the openings 4 are located at the four corners of the upper surface 2.
  • the container has an internal space surrounded by a bottom surface 1, a top surface 2 and a side surface 3 and capable of containing a culture medium.
  • the length L1 of one side of the bottom surface 1 is particularly preferably 23 to 28 cm.
  • the height L2 of the container is particularly preferably 2.5-3 cm.
  • the height L3 of the opening 4 is particularly preferably 2.5-3 cm.
  • a diameter L4 of the opening 4 is particularly preferably 3 cm.
  • FIG. 14 and 15 show the front view (14A), rear view (14B), top view (14C), bottom view (14D), right side view (14E), left side view (14F), and cross section A-A of the first container.
  • Figure (14G) perspective view (14H), front view with screw cap removed (15A), rear view with screw cap removed (15B), plan view with screw cap removed (15C), Bottom view with the screw cap removed (15D), right side view with the screw cap removed (15E), left side view with the screw cap removed (15F), cross section A-A with the screw cap removed
  • a view (15H), a perspective view (15I) with the screw cap removed, and a reference perspective view (15J) showing a transparent state with the screw cap removed are shown.
  • the first container can be used by stacking multiple sheets.
  • FIG. 16 shows a state in which four first containers are stacked. Each container is stacked on top of each other at an angle of 45 degrees. By stacking the containers while shifting them at an angle of 45 degrees, the bulkiness corresponding to the height of the opening 4 when stacking the containers can be eliminated, and space can be saved. For example, if two containers with sides 3 of 2.5 cm and openings 4 of 2.5 cm (that is, the total height of sides 3 and openings 4 is 5 cm) are placed on top of each other without shifting by 45 degrees, the two containers will have a width of 10 cm. The height is 7.5 cm when shifted by 45 degrees. In this case, it is possible to save space by 2.5 cm.
  • the length L5 of the stacked containers may be calculated by L1 ⁇ 2, the length of one side of the bottom surface 1 of the container. Furthermore, the four corners of the upper container can be fitted between the openings 4 of the lower container, and the stacked containers can be prevented from slipping and collapsing due to rocking during culturing.
  • FIG. 17 shows a front view of a state in which four first containers are stacked.
  • FIG. 18 shows an image photograph of a state in which two first containers are stacked.
  • Fig. 19 shows the second container as an example of the container.
  • the container has a square bottom 101 and top 102 shape.
  • Side 103 has a rectangular shape.
  • the side has one opening 104 .
  • the opening 104 has a port inside and a screw cap 105 is installed to cover it.
  • the opening 104 is located on the side surface 103 and at one corner on the top surface 102 side. This makes decanting easier.
  • the inside of the container has an internal space surrounded by a bottom surface 101, a top surface 102 and a side surface 103 and capable of containing a culture medium.
  • the second container can be set up sideways when changing the medium on the clean bench, making it easy to work with multiple plates at the same time.
  • a side length L101 of the bottom surface 101 is particularly preferably 23 to 40 cm.
  • the height L102 of the container is particularly preferably 5-6 cm.
  • the height L103 of the opening 104 is particularly preferably 2.5-3 cm.
  • a diameter L104 of the opening 104 is particularly preferably 3 cm.
  • a distance L105 from the lower end of the opening 104 to the bottom surface 101 on the side surface 103 of the container is particularly preferably 2.5 to 3 cm. Alternatively, the distance L105 may be determined by subtracting the length 104 of the diameter of the opening 104 from the container height L102.
  • the second container can be used by stacking multiple sheets.
  • FIG. 20 shows a state in which four second containers are stacked. Since the second container has the opening 104 provided on the side surface 103, when the containers are stacked, the containers can be stacked as they are, and a large culture area can be secured.
  • the number of containers may be, for example, 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, or 50. or within the range of two values. Large scale cultures can be performed by stacking many vessels.
  • An embodiment of the present invention is a culture vessel comprising a bottom surface, a top surface and side surfaces, the bottom surface being square shaped and the top surface comprising an opening.
  • the top surface may be square-shaped and the openings may be located at the corners (eg, one to four corners) of the top surface.
  • the opening may form a protrusion.
  • Another embodiment is a culture vessel comprising a bottom surface, a top surface and a side surface, the bottom surface being rectangular in shape and the side surface comprising an opening on the top side.
  • the top surface may be square shaped.
  • the openings may be located at corners (eg, one or two corners) on the top side of the side surface.
  • the opening may form a convexity on the surface.
  • a culture vessel which is a floating culture vessel comprising a medium containing portion, a lid, and a bottom surface of the medium containing portion having a square shape.
  • the medium containing portion may have a bottom surface and side surfaces.
  • the medium containing portion may have four sides.
  • the lid may have a square shape.
  • Another embodiment is a multi-vessel having a configuration in which multiple culture vessels are stacked. The multi-vessel is excellent in operability and suitable for large-scale culture because operations such as medium injection and medium exchange can be performed for each vessel.
  • the number of containers may be, for example, 2, 3, 4, 5, 6, 8, 10, 20, 30, 40, or 50, any value greater than or equal to any two values. may be within the range.
  • the culture vessels may be overlapped with the central axis of each vessel at the same position, while the entire vessel is shifted at an angle of 45 degrees with respect to each other.
  • the central axis may be a line extending in a direction perpendicular to the horizontal plane from the point where the diagonal lines of the quadrangular bottom surface intersect as the center.
  • the state in which the angle is 45 degrees to each other is obtained by stacking multiple containers without shifting the angle, turning the even-numbered container from the bottom 45 degrees around the central axis, and then placing the odd-numbered container from the bottom. and the bottom surface of even-numbered containers from the bottom are brought into contact with each other.
  • Another embodiment is a multiple container with a second container above the first container.
  • Another embodiment is a multiple vessel in which a plurality of culture vessels are stacked, and the top surface of the bottom culture vessel is in contact with the bottom surface of another culture vessel.
  • the culture vessel is preferably a vessel used for horizontally rotating cell culture (horizontal rotating cell culture vessel).
  • the apparatus may be a cell culture apparatus including a medium containing section and a rotation section for horizontally rotating the medium containing section, and the medium containing section having a square bottom shape.
  • the medium containing section may contain the medium and the cells.
  • the culture medium containing part may have a bottom surface, a side surface, and an upper surface, and may have a closed space inside.
  • the medium container may have an opening.
  • the opening may have a structure (for example, a cylindrical structure) that communicates the space inside the culture medium container with the space outside the container.
  • a biological sample (such as cells) or a culture medium can be injected from the space outside the container into the space inside the container through the opening.
  • the culture medium container may be a culture vessel.
  • the rotating portion may be configured to rotate the medium containing portion.
  • the rotating part may have a platform on which the medium containing part can be placed.
  • the platform may be positioned parallel to the horizontal plane.
  • the platform and the culture vessel may contact each other in a direction perpendicular to the horizontal plane.
  • the pedestal may be swiveled with the container on it.
  • One or a plurality of culture medium storage units may be installed on the platform.
  • the plurality may be, for example, 2, 4, 6, 8, 10, 20, 30, 40, 50, or 100, any value greater than or equal to any two of these values. There may be.
  • a plurality of medium storage units on the platform may be arranged without stacking or may be stacked.
  • speed of rotation of the rotating portion, the radius of gyration, etc. any of the numerical values and configurations listed in the above-described embodiments can be adopted.
  • One embodiment of the present invention is a cell culture device comprising a container and a rotating device.
  • the container may have a square bottom shape.
  • the rotating device may be a horizontal rotating device or a pivoting device.
  • the rotator may have a platform on which the container is placed.
  • An embodiment of the present invention is a cell culture vessel that contains a suspension culture medium (eg, liquid medium) and has a square bottom shape.
  • a cell culture vessel which has a square bottom shape, accommodates floating cells, and the floating cells oscillate as the vessel horizontally rotates and oscillates. is.
  • Another embodiment is a method for culturing cells, including the step of horizontally rotating a container containing cells and a culture medium, wherein the bottom shape of the container is square.
  • Another embodiment is the use of a vessel with a square bottom shape in cell culture (eg, horizontal rotary culture).
  • Another embodiment is a method for producing cells, comprising rotating a container containing cells and a medium, wherein the bottom shape of the container is square.
  • Another embodiment is a method of producing cells, comprising horizontally rotating a container containing cells and medium.
  • Another embodiment is a method for producing a cell suspension, comprising the step of horizontally rotating a container containing cells and a culture medium, wherein the bottom shape of the container is square.
  • the cell culture method in which a rectangular container is horizontally rotated has the characteristic that the cells do not gather in the center and are difficult to adhere. Therefore, for example, a method of producing a cell sheet while culturing cells in a vessel may be excluded from this culture method.
  • An embodiment of the present invention is a container having a bottom, a side and a top, comprising four sides (first to fourth sides), the first side and the third side being in parallel relationship, The container, wherein the second side and the fourth side are in parallel relationship.
  • the first side and the second side may be in contact, and the first side and the fourth side may be in contact.
  • the second side and the third side may be in contact, and the third side and the fourth side may be in contact.
  • the four sides may be in contact with the bottom surface at the bottom and the top surface at the top.
  • One embodiment of the present invention is a method for dispersing cells, which includes the step of horizontally rotating a container containing cells and a culture medium, and the bottom surface of the container has a square shape. Dispersion of cells facilitates the distribution of nutrients to the cells. In addition, shear stress is reduced and cell viability is improved.
  • One embodiment of the present invention is a method for enzymatic treatment of cells, which includes the step of horizontally rotating a container containing cells and an enzyme solution, and the bottom shape of the container is square. According to this method, an excellent enzymatic treatment can be achieved from at least one viewpoint of improving the viable cell rate after treatment, improving the number of cells after treatment, or reducing shear stress during treatment.
  • the enzyme solution may be, for example, a solution containing a proteolytic enzyme.
  • This solution includes, for example, a solution that disperses cell clumps or adherent cells into single cells.
  • This solution includes, for example, TrypLE Select (Gibco 12563-011), trypsin solution, accutase solution, collagenase solution.
  • the cells contained together with the enzyme solution may be, for example, cell aggregates or cells adhered to plastic material.
  • This method includes, for example, a step of suspension culture of cells, a step of contacting the cells with an enzyme solution, a step of preparing a suspension containing the cells and the enzyme solution, or a step of replacing the medium in the container with the enzyme solution. It's okay.
  • the content of enzyme in the suspension may be an effective amount for cell dispersion.
  • the enzymatic treatment includes, for example, dispersing treatment of cells.
  • the embodiment of the enzyme treatment method described above can employ any of the numerical values, configurations (eg, cells, etc.), and steps listed in the embodiment of the cell production method described above.
  • cells include stem cells (eg, iPS cells or mesenchymal stem cells).
  • One embodiment of the present invention is an apparatus for treating cells with enzymes, which includes a medium container and a rotating part that horizontally rotates the medium container, and the medium container has a square bottom shape.
  • An enzyme treatment device is provided. Using this device, excellent enzymatic treatment can be achieved from at least one viewpoint of improving post-treatment viability, improving post-treatment cell number, or reducing shear stress during treatment.
  • This embodiment of the cell enzyme treatment device can adopt any of the numerical values and configurations listed in the above-described embodiments of the cell culture device or the embodiment of the enzyme treatment method.
  • One embodiment of the present invention is a horizontal rotary cell enzyme treatment vessel comprising a bottom, a top and a side, the bottom being square-shaped and the top comprising 1-4 openings.
  • a cellular enzyme treatment vessel is provided. This vessel is used for enzymatic treatment of cells with the vessel rotated horizontally. By using this container, an excellent enzymatic treatment can be achieved in terms of at least one of improving the post-treatment viable cell rate, improving the post-treatment cell count, and reducing shear stress during treatment.
  • This embodiment of the horizontally rotating cell enzymatic treatment container can employ any of the numerical values and configurations listed in the above embodiments of the container or the enzymatic treatment method.
  • iPS cells (253G1 strain) were adherently cultured in a 10 cm dish coated with iMatrix-511 (Nippi) in Essential 8 medium (Thermo Fisher), and then cultured in 0.5 mM EDTA solution. iPS cells detached after being treated with for 10 minutes were used in the experiment.
  • the exfoliated iPS cells were suspended in Essential 8 medium to a cell concentration of 7x10e4/ml and placed in a 6 cm diameter (20 cm 2 area) circular dish (Falcon), a 10 cm diameter (50 cm 2 area) circular dish (Falcon), A circular dish (Falcon) with a diameter of 15 cm (area of 150 cm 2 ) and a square dish (Sumitomo Bakelite, product number MS-12450) with a side of 22 cm (area of 500 cm 2 ) were each filled with a liquid volume of 0.4 ml/cm 2 . 8 ml, 20 ml, 60 ml, and 200 ml of cell suspension were added, and static culture or rotary culture was performed in an incubator at 37° C.
  • Spinning culture was performed using an incubator internal shaker (Optima, OS-762RC) at 30, 50, and 60 rpm (circling diameter: 25 mm). After culturing for a total of 4 days, the floating iPS cell clusters were dispersed with a trypsin solution and counted. ) was calculated.
  • plastic beads and mesenchymal stem cells (UE6E7T-3 cells JCRB1136).
  • Plastic beads (125-212 ⁇ m diameter, Corning 3772, similar size and density to iPS spheroids) were added to the medium at a volume of 1 gram/50 ml and mesenchymal stem cells at a volume of 2 ⁇ 10e6 cells/50 ml.
  • 60 ml of the medium containing beads and mesenchymal stem cells was added to a circular dish (15 cm in diameter) and 200 ml to a square dish (1 side of 22 cm), and cultured in a 37° C. incubator for 2 days. After the beads and mesenchymal stem cells adhered, the image was taken while rotating culture at 30 rpm with a shaker (Optima, OS-762RC).
  • the same plastic beads and mesenchymal stem cells containing 10% FBS were added to compare the shape of the container during enzyme treatment.
  • Culture was performed in DMEM medium. 200 ml of a medium containing 1.7 g of beads and 3 ⁇ 10e6 cells of mesenchymal stem cells was added to a square dish (22 cm per side), and cultured for 7 days with a shaker rotating at 50 rpm to proliferate the cells.
  • each dish containing the mesenchymal stem cell clusters was spin-cultured at 80 rpm for 30 minutes to disperse the mesenchymal stem cell clusters into single cells, which were then counted. was calculated.
  • the enzyme solution may be trypsin or accutase solution.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un excellent procédé de production cellulaire.　L'utilisation est faite d'un procédé de production cellulaire comprenant une étape consistant à faire tourner horizontalement un récipient contenant des cellules et un milieu, le fond du récipient étant de forme carrée. En variante, il est possible d'utiliser un dispositif de culture cellulaire comprenant une partie de logement du milieu et une partie rotative pour faire tourner horizontalement la partie de logement du milieu, le fond de la partie de logement du milieu étant de forme carrée.
PCT/JP2022/006663 2021-02-19 2022-02-18 Procédé de production cellulaire WO2022176980A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021025619A JP2024060111A (ja) 2021-02-19 2021-02-19 細胞の生産方法
JP2021-025619 2021-02-19

Publications (1)

Publication Number Publication Date
WO2022176980A1 true WO2022176980A1 (fr) 2022-08-25

Family

ID=82930716

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/006663 WO2022176980A1 (fr) 2021-02-19 2022-02-18 Procédé de production cellulaire

Country Status (2)

Country Link
JP (1) JP2024060111A (fr)
WO (1) WO2022176980A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008212049A (ja) * 2007-03-02 2008-09-18 Fujimori Kogyo Co Ltd 培養袋及び培養装置
JP2015509701A (ja) * 2011-12-02 2015-04-02 アーメイゲン・テクノロジーズ・インコーポレイテッドＡｒｍａｇｅｎ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ． Ｃｎｓにおけるアリールスルファターゼａ活性を増加するための方法および組成物
WO2017094451A1 (fr) * 2015-12-04 2017-06-08 公立大学法人大阪府立大学 Récipient de culture cellulaire et cellule à échantillon pour observation
US20180147552A1 (en) * 2016-11-28 2018-05-31 Sarfaraz K. Niazi Zero gravity process device
JP2019198850A (ja) * 2018-05-18 2019-11-21 藤森工業株式会社 撹拌容器および撹拌方法
JP2021027821A (ja) * 2019-08-09 2021-02-25 宇部興産株式会社 細胞培養装置及びそれを使用した細胞培養方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008212049A (ja) * 2007-03-02 2008-09-18 Fujimori Kogyo Co Ltd 培養袋及び培養装置
JP2015509701A (ja) * 2011-12-02 2015-04-02 アーメイゲン・テクノロジーズ・インコーポレイテッドＡｒｍａｇｅｎ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ． Ｃｎｓにおけるアリールスルファターゼａ活性を増加するための方法および組成物
WO2017094451A1 (fr) * 2015-12-04 2017-06-08 公立大学法人大阪府立大学 Récipient de culture cellulaire et cellule à échantillon pour observation
US20180147552A1 (en) * 2016-11-28 2018-05-31 Sarfaraz K. Niazi Zero gravity process device
JP2019198850A (ja) * 2018-05-18 2019-11-21 藤森工業株式会社 撹拌容器および撹拌方法
JP2021027821A (ja) * 2019-08-09 2021-02-25 宇部興産株式会社 細胞培養装置及びそれを使用した細胞培養方法

Also Published As

Publication number Publication date
JP2024060111A (ja) 2024-05-02

Similar Documents

Publication Publication Date Title
JP6892486B2 (ja) 培養方法
JP2022127556A (ja) 細胞の生産方法
Ouyang et al. Three-dimensional bioprinting of embryonic stem cells directs highly uniform embryoid body formation
MX2015002819A (es) Dispositivos y metodos para el cultivo de celulas.
Powers et al. Development and optimization of AAV hFIX particles by transient transfection in an iCELLis® fixed-bed bioreactor
WO2019014627A1 (fr) Éléments de manipulation pour récipient de culture cellulaire à microcavités
EP3652294A1 (fr) Récipient de culture cellulaire
JP5558560B2 (ja) バイオリアクターシステム
JP7068285B2 (ja) 細胞培養容器
Nogueira et al. Single-use bioreactors for human pluripotent and adult stem cells: towards regenerative medicine applications
CN204644371U (zh) 一种集成式多细胞非接触共培养装置
WO2022176980A1 (fr) Procédé de production cellulaire
CN111139218A (zh) 用诱导多能干细胞或胚胎干细胞快速高效制备拟胚体的方法
US9518249B2 (en) Cell carrier, associated methods for making cell carrier and culturing cells using the same
US20160046898A1 (en) Cell growth macrocarriers for bioreactors
WO2019055448A1 (fr) Dispositifs de culture en suspension et systèmes et procédés associés
Iwai et al. Induction of cell self‐organization on weakly positively charged surfaces prepared by the deposition of polyion complex nanoparticles of thermoresponsive, zwitterionic copolymers
US11584906B2 (en) Cell culture vessel for 3D culture and methods of culturing 3D cells
US20100028992A1 (en) Cell culture device
JP2014204671A (ja) 細胞培養担体
CN206375920U (zh) 一种用于培养细胞的培养皿
CN213977735U (zh) 一种软骨膜片自分泌因子保留培养皿
CN214654882U (zh) 一种简易可拆卸双室培养皿
US20200347334A1 (en) Macrocarriers for cell growth in bioreactors
CN209778891U (zh) 一种新型间充质干细胞培养容器

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22756297

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22756297

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP