WO2015105029A1 - Récipient de culture de cellules, système de sous-culture de cellules et procédé de sous-culture de cellules - Google Patents

Récipient de culture de cellules, système de sous-culture de cellules et procédé de sous-culture de cellules Download PDF

Info

Publication number
WO2015105029A1
WO2015105029A1 PCT/JP2014/084597 JP2014084597W WO2015105029A1 WO 2015105029 A1 WO2015105029 A1 WO 2015105029A1 JP 2014084597 W JP2014084597 W JP 2014084597W WO 2015105029 A1 WO2015105029 A1 WO 2015105029A1
Authority
WO
WIPO (PCT)
Prior art keywords
substrate
cell
cell culture
cells
culture
Prior art date
Application number
PCT/JP2014/084597
Other languages
English (en)
Japanese (ja)
Inventor
加川　健一
祐介 依田
成則 尾▲崎▼
Original Assignee
東京エレクトロン株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2014002626A external-priority patent/JP2017046591A/ja
Priority claimed from JP2014002636A external-priority patent/JP2017046592A/ja
Application filed by 東京エレクトロン株式会社 filed Critical 東京エレクトロン株式会社
Publication of WO2015105029A1 publication Critical patent/WO2015105029A1/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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps

Definitions

  • the present invention relates to a cell culture container.
  • the present invention also relates to a cell subculture method using the cell culture container of the present invention.
  • the present invention further relates to a cell subculture system provided with the cell culture container of the present invention.
  • the present invention further relates to a cell culture system provided with the cell culture container of the present invention.
  • the present invention further relates to a method for detaching cells cultured in the cell culture system of the present invention.
  • the cells are detached from the adhesion surface of the culture container using an enzyme treatment or a cell scraper, and subcultured to a culture container containing a fresh medium.
  • pluripotent stem cells are killed when dissociated into single cells (Non-patent Document 1), and it is essential to pass in the state of a cell mass during passage. It has become. Therefore, in the passage of pluripotent stem cells, when detaching the cells from the culture vessel, the pluripotent stem cells are detached as colonies, crushed into a cell mass of an appropriate size by pipetting, etc., and then new Seeded in culture vessel.
  • Non-Patent Document 2 a technique for obtaining a cell cluster of a certain size from pluripotent stem cells.
  • the cell clump obtained by these methods has an embryoid body structure, and the embryoid body is formed when cells are differentiated. In particular, when an embryoid body is formed in a cell and cultured, the cell starts to differentiate.
  • This technique is known as one of general methods for examining the pluripotency of a cell. Therefore, in the method such as Non-cited Document 2, it is considered that the obtained cell clump is changed to a state that is easily differentiated. It is also known that the formation of embryoid bodies is useful as a pretreatment for differentiating cells (Non-patent Documents 2 and 3).
  • pluripotent stem cells are also known to be easily differentiated, and maintaining the undifferentiated state of the cells is the most important issue in subculture of pluripotent stem cells.
  • Pluripotent stem cells are particularly difficult to return to an undifferentiated state once differentiation has begun. Therefore, pluripotent stem cells should not be changed to a state in which cells are easily differentiated in maintenance culture.
  • the open culture is a culture using a normal dish in which the medium is exchanged and passaged by opening the lid of the dish. Open culture is often used for research cell culture because it is inexpensive and easy to operate. However, there is a high possibility of contamination with bacteria and cells. There are many safety issues such as high risk of infection for workers, and it is difficult to say that it is a perfect equipment for cell culture for treatment.
  • closed cell culture has been attempted in cell culture for medical use.
  • closed culture a closed culture vessel having a minimum opening is used, and a device for greatly reducing the incidence of contamination and the risk of infection has been made (Patent Document 1).
  • a cell detachment solution and a device that detaches cells by vibration As a means for detaching cultured cells adhering to the inner wall surface of the culture container from the inner wall surface, a cell detachment solution and a device that detaches cells by vibration (Patent Document 2) and a cell detachment solution A device that collects detached cells by tilting the container (Patent Document 3) has been developed.
  • pluripotent stem cells such as embryonic stem cells (ES cells) and inducible pluripotent stem cells (iPS cells)
  • ES cells embryonic stem cells
  • iPS cells inducible pluripotent stem cells
  • cells that have started differentiation in the middle of the culture have adverse effects on surrounding cells. And should be promptly removed from the culture vessel.
  • cells that have started to differentiate should be removed promptly in order to increase the yield and purity of undifferentiated cells.
  • the cells that have started differentiation are removed by, for example, opening the dish lid, inserting an aspirator or micropipette into the dish, and sucking and removing the cells that have started differentiation (non-patented). Reference 4).
  • the culture system is in contact with the outside world, there is a concern about the occurrence of contamination in the open culture.
  • closed culture of pluripotent stem cells has been attempted. In closed culture, the possibility of contamination is low, but an aspirator cannot be inserted into the culture vessel, and it is difficult to selectively remove cells that have started differentiation during the culture
  • the present invention relates to a cell culture container for efficient and homogeneous subculture of cells, particularly pluripotent stem cells, a cell subculture method using the cell culture container, and a cell using the culture container A subculture system is provided.
  • the present invention also provides a closed cell culture container for efficiently or selectively detaching cells from the cell adhesion surface, a cell culture system provided with the cell culture container, and a cell cultured in the cell culture system.
  • a peeling method is provided.
  • a cell culture container One substrate, The other substrate disposed opposite the one substrate; A side wall that is interposed between one substrate and the other substrate to form a culture chamber having a cell adhesion surface formed by at least one of the one substrate and the other substrate and a flow path; With One substrate is made of stretchable material, Cell culture container.
  • One substrate is made of stretchable material, Cell culture container.
  • a cell subculture system The cell culture vessel according to any one of (1) to (4) above; Provided outside the one substrate of the cell culture container, and having an enclosure having a concavo-convex shape on the facing surface facing the one substrate, A vacuum source is connected to the opposing surface of the enclosure, and by operating this vacuum source, the opposing surface having the uneven shape of the enclosure is evacuated to adsorb one substrate to the opposing surface having the uneven shape of the enclosure. To form an uneven shape on one of the substrates, Cell subculture system. (6) The subculture system according to (5), wherein the vacuum source is connected to the bottom surface of each concave portion on the opposing surface.
  • a cell subculture system The cell culture vessel according to any one of (1) to (4) above; Provided outside the one substrate of the cell culture container, and an enclosure having a plurality of protrusions on the facing surface facing the one substrate, A vacuum source is connected to the opposing surface of the enclosure, and by operating this vacuum source, the opposing surface having the uneven shape of the enclosure is evacuated to form an uneven shape with protrusions on one substrate.
  • a vacuum source is connected to the opposing surface of the enclosure, and by operating this vacuum source, the opposing surface having the uneven shape of the enclosure is evacuated to form an uneven shape with protrusions on one substrate.
  • a cell culture container One substrate, The other substrate disposed opposite the one substrate; A side wall that is interposed between one substrate and the other substrate to form a culture chamber having a cell adhesion surface formed by at least one of the one substrate and the other substrate and a flow path;
  • One substrate consists of a rigid frame part that forms a plurality of openings, and a stretchable part that extends into each opening of the frame, A cell culture container in which when a negative pressure is applied to one substrate from the outside, an expansion / contraction portion in the frame of one substrate extends to form an uneven shape on one substrate.
  • a cell culture container One substrate, The other substrate disposed opposite the one substrate; A side wall that is interposed between one substrate and the other substrate to form a culture chamber having a cell adhesion surface formed by at least one of the one substrate and the other substrate and a flow path;
  • One substrate is composed of a plurality of hard portions and a stretchable portion extending between the hard portions, A cell culture container in which, when a negative pressure is applied to one substrate from the outside, a stretchable portion of one substrate extends to form an uneven shape on one substrate.
  • a cell culture system The cell culture container according to (8) or (9) above, The outside of the one substrate of the cell culture container, and a enclosure that forms a vacuum chamber between the one substrate, A vacuum source is connected to the vacuum chamber, and by operating this vacuum source, the vacuum chamber is evacuated to form an uneven shape on one substrate.
  • Cell subculture system (11) The cell subculture system according to any one of (5) to (7) and (10) above, wherein the enclosure is removable from the cell culture container and is selected. A desired enclosure can be attached to the cell culture vessel, Cell subculture system.
  • a cell passage method using the cell culture vessel according to any one of (1) to (4), (8) and (9) above (A) a step of dispersing cells during passage; (B) injecting the dispersed cells into the culture chamber of the cell culture container; (C) using the method according to any of (13) to (18) above, forming a concavo-convex shape on one substrate facing the culture chamber of the cell culture vessel; (D) forming a cell clump on the cells seeded in the concavo-convex recess formed on one substrate. (20) The method according to (19), further comprising the step of (E) inverting the cell culture container upside down and seeding the formed cell mass on the other substrate.
  • step (E) Release the evacuation of the vacuum chamber to eliminate the uneven shape formed on one substrate, and peel off the cell conglomerate formed in step (D) from one substrate, Supplying a gas to the vacuum chamber and pushing one substrate into the culture chamber to peel off the cell conglomerate formed in step (D) from one substrate, or By repeating the operation of evacuation of the vacuum chamber and the operation of supplying gas to the vacuum chamber and applying vibration to one of the substrates, the cell conglomerate formed in the step (D) is peeled from the one substrate.
  • a closed cell culture vessel A first surface made of elastic material; A second surface disposed opposite the first surface; A sidewall disposed between the first surface and the second surface; A culture chamber serving also as a flow path of the solution defined by the first surface, the second surface, and the side wall; A solution inlet; A closed cell culture vessel equipped with (24) The closed-system cell culture container according to (23), wherein the first surface is deformable into a concave shape so as to form a cell clump. (25) The closed cell culture vessel according to (23) or (24) above, wherein the second surface has cell adhesiveness.
  • a cell culture system comprising a cell culture container and a pressing body, Cell culture container One substrate, The other substrate disposed opposite the one substrate; A culture chamber having a cell adhesion surface formed by at least one of the one substrate and the other substrate and a side wall forming a flow path, interposed between the one substrate and the other substrate; One substrate is made of elastic material, The pressing body is a pressing body that is installed outside one substrate, presses one substrate toward the culture chamber, and retracts into the culture chamber. Cell culture system.
  • a cell culture system comprising a cell culture container and a pressing body, Cell culture container A first substrate; A second substrate disposed opposite the first substrate; A side wall that is interposed between the first substrate and the second substrate to form a culture chamber having a cell adhesion surface formed by at least one of the first substrate and the second substrate, and a flow path;
  • the first substrate is made of elastic material
  • the pressing body is a pressing body that is installed outside the first substrate, presses the first substrate toward the culture chamber, and retracts into the culture chamber.
  • Cell culture system. The cell culture system according to any one of (26) to (28), wherein the stretchable material is transparent.
  • the pressing body has a pressing surface corresponding to the planar shape of the entire culture chamber, whereby one substrate corresponding to the planar shape of the entire culture chamber can be pressed by the pressing body (26 ) To (31).
  • the cell culture system according to any one of (26) to (31), wherein the pressing body is smaller than the planar shape of the entire culture chamber and has a pressing surface of 0.12 mm 2 to 9 mm 2 .
  • a cell culture container One substrate, The other substrate disposed opposite the one substrate; A sidewall interposed between one substrate and the other substrate to form a culture chamber having a cell adhesion surface formed by at least one of the one substrate and the other substrate; An inlet for flowing liquid into the culture chamber; An outlet for draining liquid from the culture chamber; A sub-side wall standing up from the other substrate in the culture chamber and having a lower height than the side wall, and forming a groove extending from the inlet to the outlet in the culture chamber; With One substrate is made of an elastic material, and one substrate is separated from the sub-side wall to form an integral space in the culture chamber, When one of the substrates comes into contact with the sub-side wall, a channel communicating with the inlet and the outlet is formed by the groove.
  • a cell culture system The cell culture vessel according to (40) above, A first pressing body installed outside one substrate of the cell culture container; With The first pressing body presses one substrate toward the culture chamber to contact the sub-side wall, Cell culture system.
  • the first pressing body has a pressing surface corresponding to the planar shape of the culture chamber, and can thereby press a portion of the one substrate corresponding to the pressing surface toward the culture chamber.
  • a second pressing body is further provided,
  • the second pressing body has a pressing surface that can enter the groove portion of the cell culture container, and thereby, a portion corresponding to the pressing surface of one substrate is pressed toward the culture chamber to enter the groove portion.
  • the cell culture system according to any one of (41) to (43), which can be pushed in.
  • the cell according to (44) or (45) further comprising second pressing body driving means for driving the second pressing body to scrape off the cells adhered to the cell adhesion surface with one substrate. Culture system.
  • Method. A method for peeling cells cultured in the cell culture system according to any one of (41) to (46), A method comprising the step of pressing one substrate toward the culture chamber by the first pressing body to contact the sub-side wall and then peeling the cells by feeding the cell recovery solution to the culture chamber.
  • a method for separating cells cultured in the cell culture system according to any one of (44) to (46), A step of pressing the one substrate toward the culture chamber by the second pressing body and pushing it into the groove, driving the second pressing body driving means to detach the cells by scraping the cells with the one substrate.
  • Comprising a method. (50) The method according to (48), further comprising feeding a cell detachment solution to weaken cell adhesion before feeding the cell recovery solution.
  • the method according to (48) further comprising applying high-frequency vibration to the cell adhesion surface of the other substrate to weaken cell adhesion before feeding the cell recovery solution.
  • FIG. 1 is an upper plan view of the cell culture container in the first embodiment of the present invention.
  • FIG. 2 is a side sectional view of the cell culture container of FIG.
  • FIG. 3 is a side cross-sectional view of the cell culture container of FIG.
  • FIG. 4A is a plan view of an enclosure used in the cell subculture system in the first embodiment of the present invention.
  • 4B is a side cross-sectional view taken along the line AA in FIG. 4A.
  • FIG. 4C is a partial schematic cross-sectional view showing a state in which the cell culture container and the enclosure are brought into contact with each other in the cell subculture system of the first embodiment.
  • FIG. 1 is an upper plan view of the cell culture container in the first embodiment of the present invention.
  • FIG. 2 is a side sectional view of the cell culture container of FIG.
  • FIG. 3 is a side cross-sectional view of the cell culture container of FIG.
  • FIG. 4A is a plan view of an enclosure used in the cell subculture system in
  • FIG. 4D is an upper plan view showing a state in which one substrate is adsorbed to the concavo-convex shape of the surrounding body and the concavo-convex shape is formed on one substrate in the cell subculture system of the first embodiment.
  • 4E is a partial schematic cross-sectional view taken along the line AA of FIG. 4D.
  • FIG. 5A is an upper plan view of one substrate of the cell culture container in the third embodiment.
  • FIG. 5B is a side cross-sectional view taken along the line AA in FIG. 5A.
  • FIG. 5C is an upper plan view of one substrate of the cell culture container in the modification of the third embodiment.
  • 5D is a side cross-sectional view taken along the line BB of FIG. 5C.
  • FIG. 5E is a partial schematic cross-section showing a state in which a negative pressure is applied to one substrate of a cell culture container from the outside to form an uneven shape on one substrate in the cell subculture system of the third embodiment.
  • FIG. FIG. 5F is a side cross-sectional view of the cell culture container according to the third embodiment, taken along a cross section corresponding to the AA cross section of FIG.
  • FIG. 6A is an upper plan view of one substrate of a cell culture container according to another modification of the third embodiment.
  • 6B is a side cross-sectional view taken along the line AA in FIG. 6A.
  • FIG. 6C is a side cross-sectional view corresponding to FIG. 6B of one substrate of the cell culture container in still another modified example of the third embodiment.
  • 6D shows a cell subculture system having a cell culture container according to another modification of the third embodiment, in which negative pressure is applied to one substrate of the cell culture container from the outside, and the one substrate is uneven. It is a partial schematic sectional drawing which shows the state in which the shape was formed.
  • 6E is a side cross-sectional view of a cell culture container according to another modification of the third embodiment, taken along a line AA in FIG.
  • FIG. 7A is an upper plan view of an enclosure used in the cell subculture system in the second embodiment of the present invention.
  • FIG. 7B is a side cross-sectional view taken along the line AA in FIG. 7A.
  • FIG. 7C shows a cell passage culture system according to the second embodiment, in which one substrate and the enclosure are brought into contact so as to form a vacuum chamber between the one substrate and the enclosure, and then the vacuum source
  • FIG. 6 is an upper plan view showing a state in which a concavo-convex shape is formed on one substrate by protrusions by operating
  • FIG. 7D is a partial schematic cross-sectional view taken along the line AA in FIG. 7C.
  • FIG. 8A is a partial schematic cross-sectional view showing a method for subculturing cells using the cell subculture system in the second embodiment, in a state where the cell culture container is filled with a cell suspension. It is a partial schematic sectional drawing shown.
  • FIG. 6 is an upper plan view showing a state in which a concavo-convex shape is formed on one substrate by protrusions by operating
  • FIG. 7D is a partial schematic cross-sectional view taken along the line AA in FIG. 7C.
  • FIG. 8A is
  • FIG. 8B is a partial schematic cross-sectional view showing a method for subculturing cells using the cell subculture system in the second embodiment, and evacuating the vacuum chamber to one substrate of the cell culture container. It is a partial schematic sectional drawing which shows the state in which many microwells were formed.
  • FIG. 8C is a partial schematic cross-sectional view showing a method for subculturing cells using the cell subculture system according to the second embodiment, in which cells are driven by rotating a fixture by driving a rotation mechanism. It is a partial schematic sectional drawing which shows the state which inverted the culture container and the enclosure upside down, and seed
  • FIG. 8D is a partial schematic cross-sectional view showing a method for subculturing cells using the cell subculture system in the second embodiment, in which some cell clumps still adhere to one substrate.
  • the evacuation of the vacuum chamber is released to eliminate the uneven shape of the one substrate, thereby dropping the cell clump attached to one substrate onto the other substrate.
  • FIG. 8E is a partial schematic cross-sectional view showing a method for subculturing cells using the cell subculture system in the second embodiment, and after seeding a cell conglomerate on the other substrate, It is a partial schematic sectional drawing which shows the state which culture
  • FIG. 9A is a partial schematic cross-sectional view illustrating a process of dissociating a cell clump from one substrate and seeding on the other substrate in FIG. 8C, and still remains after the uneven shape of one substrate is eliminated. It is a partial schematic sectional drawing which shows the state which the cell clump of a part has adhere
  • FIG. 9B is a partial schematic cross-sectional view illustrating a process of dissociating a cell clump from one substrate and seeding on the other substrate in FIG. 8C, and culturing one substrate by supplying gas to the vacuum chamber It is a partial schematic sectional drawing explaining the method of pushing into a chamber and thereby dissociating a cell clump from one substrate and dropping it on the other substrate.
  • FIG. 9C is a partial schematic cross-sectional view for explaining the process of dissociating the cell clump from one substrate and seeding the other substrate in FIG. 8C, and evacuating the vacuum chamber and supplying the gas to the vacuum chamber
  • FIG. 5 is a partial schematic cross-sectional view for explaining a method of repeating the above, whereby a cell clump is dissociated from one substrate and dropped onto the other substrate.
  • FIG. 10A is a partial schematic cross-sectional view for explaining that a cell agglomeration is formed on one substrate of the cell culture container in the first embodiment, and then culturing is performed using one substrate as a cell adhesion surface as it is.
  • FIG. 10B is a partial schematic cross-sectional view for explaining that cell agglomeration is formed on one substrate of the cell culture container in the first embodiment, and then culturing is performed using one substrate as a cell adhesion surface as it is.
  • the vacuum chamber is evacuated to form a large number of microwells on one substrate of the cell culture container, and cells are seeded in the formed microwells to form a cell clump from the cells.
  • FIG. 10B is a partial schematic cross-sectional view for explaining that cell agglomeration is formed on one substrate of the cell culture container in the first embodiment, and then culturing is performed using one substrate as a cell adhesion surface as it is.
  • the vacuum chamber is evacuated to form a large number of microwells on one substrate of the cell culture container, and cells are seeded in the formed microwells to form a cell clump from the cells.
  • FIG. 10B is a partial schematic cross-sectional view for explaining that cell agglomeration is formed on
  • FIG. 10C is a partial schematic cross-sectional view for explaining that a cell agglomeration is formed on one substrate of the cell culture container in the first embodiment, and then culturing is performed using the one substrate as a cell adhesion surface as it is. Therefore, without inverting the cell culture container upside down, the vacuum chamber is released and the uneven shape formed on one substrate is eliminated, and the cells are left on the cell adhesion surface on one substrate.
  • FIG. 11A is a diagram showing the state of cells immediately after seeding AggreWell 800 with human iPS cells dispersed to a single cell level and one day later.
  • FIG. 11B is a diagram showing a cell clump obtained by seeding AggreWell 800 with human iPS cells dispersed to a single cell level.
  • FIG. 12 is a phase contrast microscopic image of cells after passage of cell clumps obtained with or without centrifugation.
  • FIG. 13 is a diagram showing a growth curve after passage of the obtained cell conglomerate.
  • FIG. 14 is a figure which shows the phase-contrast microscope image and fluorescence immunostaining image of the colony after a subculture.
  • FIG. 15 is a diagram showing the arrangement on the culture surface of the cell clumps dropped on the culture surface of the culture container.
  • FIG. 16 is a plan view of a cell culture system according to the fourth embodiment of the present invention.
  • FIG. 17 is a cross-sectional view taken along the line AA in FIG. 18 is a cross-sectional view taken along the line BB in FIG.
  • FIG. 19A is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fourth embodiment of the present invention, showing a state in which cells are cultured on the cell adhesion surface of the other substrate. It is a schematic sectional drawing.
  • FIG. 19B is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fourth embodiment, and shows a partial schematic view showing a state where one substrate is pressed by a pressing body and pushed toward the culture chamber. It is sectional drawing.
  • FIG. 19A is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fourth embodiment of the present invention, showing a state in which cells are cultured on the cell adhesion surface of the other substrate. It is a schematic sectional drawing.
  • FIG. 19B is a partial schematic
  • FIG. 19C is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fourth embodiment.
  • the pressed body is operated by the pressing body operating means, and the cells are moved through one substrate.
  • It is a partial schematic sectional drawing which shows the state which has been scraped off.
  • FIG. 19D is a partial schematic cross-sectional view showing an example of the cell culture system of the fourth embodiment and its operation, and is a partial schematic cross-sectional view showing a state where scraped cells are peeled off from the substrate and collected or removed. It is.
  • FIG. 19D is a partial schematic cross-sectional view showing an example of the cell culture system of the fourth embodiment and its operation, and is a partial schematic cross-sectional view showing a state where scraped cells are peeled off from the substrate and collected or removed. It is.
  • FIG. 20A is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fifth, seventh and eighth embodiments of the present invention, in which cells are cultured on the cell adhesion surface of the other substrate.
  • FIG. 20B is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fifth, seventh and eighth embodiments, wherein one substrate is pressed by a pressing body and pushed toward the culture chamber. It is a partial schematic sectional drawing which shows a state.
  • FIG. 20C is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fifth, seventh and eighth embodiments, in a state where one substrate is pressed by a pressing body into the culture chamber.
  • FIG. 5 is a partial schematic cross-sectional view showing a state in which the cell recovery liquid is supplied to the flow path and the culture chamber by operating the liquid supply means for supplying the cell recovery liquid.
  • FIG. 20D is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the fifth, seventh and eighth embodiments, and cells are detached from the cell adhesion surface corresponding to the pressing surface of the pressing body. It is a partial schematic sectional drawing which shows the state collect
  • FIG. 21 is a diagram showing the relationship between the shear stress generated when a viscous liquid flows in a cylinder, the width of the flow path, and the flow rate.
  • FIG. 22A is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment of the present invention, in which a high-frequency vibration generating unit is installed outside the other substrate of the cell culture container. It is a partial schematic sectional drawing which shows the state which exists.
  • FIG. 22B is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, which applies high-frequency vibration to the other substrate on which cells are cultured on the cell adhesion surface. It is a partial schematic sectional drawing which shows the state which has weakened the adhesiveness of a cell adhesion surface and a cell.
  • FIG. 22C is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, and shows a partial schematic view showing a state where one substrate is pressed by a pressing body and pushed toward the culture chamber. It is sectional drawing.
  • FIG. 22D is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, in which the cell recovery solution is placed in a state where one substrate is pressed into the culture chamber by a pressing body. It is a partial schematic sectional drawing which shows the state which operates the liquid feeding means to send liquid, and is feeding the cell collection
  • FIG. 22E is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, and shows a state where cells are peeled from the cell adhesion surface corresponding to the pressing surface of the pressing body. It is a schematic sectional drawing.
  • FIG. 22F is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, in which cells are peeled from the cell adhesion surface corresponding to the pressing surface of the pressing body, and are collected or removed. It is a partial schematic sectional drawing which shows a state.
  • FIG. 22E is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the ninth embodiment, and shows a state where cells are peeled from the cell adhesion surface corresponding to the pressing surface of the pressing body. It is a schematic sectional drawing.
  • FIG. 23A is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the sixth embodiment of the present invention, showing a state in which cells are cultured on the cell adhesion surface of the other substrate. It is a schematic sectional drawing.
  • FIG. 23B is a partial schematic cross-sectional view showing an example of the cell culture system of the sixth embodiment and its operation, and shows a state in which a part of one substrate is pressed by a pressing body and pushed toward the culture chamber. It is a partial schematic sectional drawing shown.
  • FIG. 23C is a partial schematic cross-sectional view showing an example of the cell culture system and its operation according to the sixth embodiment, in which a cell is pressed into a culture chamber by pressing a part of one substrate with a pressing body.
  • FIG. 5 is a partial schematic cross-sectional view showing a state in which a liquid feeding means for feeding a collected liquid is operated and a cell collected liquid is fed to a channel and a culture chamber.
  • FIG. 23D is a partial schematic cross-sectional view showing an example of the cell culture system of the sixth embodiment and its operation, in which cells are peeled off from a part of the cell adhesion surface corresponding to the pressing surface of the pressing body, and are collected or It is a partial schematic sectional drawing which shows the state removed.
  • FIG. 24A is a partial schematic cross-sectional view showing a modified example of the cell culture system of the fourth embodiment, in which a pressing body is arranged on the outside of one substrate and another on the outside of the other substrate.
  • FIG. 24B is a partial schematic cross-sectional view showing a modification of the cell culture system according to the fourth embodiment, and a pressing body disposed outside one substrate and a pressing body disposed outside the other substrate. It is a partial schematic sectional view which shows the state which pushed one board
  • FIG. 24C is a partial schematic cross-sectional view showing a modification of the cell culture system of the sixth and eighth embodiments.
  • FIG. 24D is a partial schematic cross-sectional view showing a modification of the cell culture system of the fifth, seventh and eighth embodiments.
  • FIG. 24E is a partial schematic cross-sectional view showing a modification of the cell culture system of the fifth, seventh and eighth embodiments.
  • FIG. 24F is a partial schematic cross-sectional view showing a modification of the cell culture system of the ninth embodiment.
  • FIG. 25A is a plan view of the cell culture container prepared in Example 3.
  • FIG. 25B is a front view of the cell culture container prepared in Example 3.
  • FIG. 25C is a side view of the cell culture container prepared in Example 3.
  • FIG. 25D is a bottom view of the cell culture container prepared in Example 3.
  • FIG. 25E is a cross-sectional view taken along line AA of FIG. 25A.
  • FIG. 25F is a sectional view taken along line BB of FIG. 25A.
  • FIG. 26A is a diagram showing the results of a cell detachment experiment using the iPS cells cultured in Example 4 and the cell culture system of the present invention, and is a diagram showing a phase contrast microscopic image of the cells immediately after the culture.
  • FIG. 26B is a diagram showing the results of a cell detachment experiment using the iPS cells cultured in Example 4 and the cell culture system of the present invention, and is a diagram showing a phase contrast microscopic image of the cells after treatment with the cell detachment solution. is there.
  • FIG. 26C is a diagram showing the results of cell detachment experiments using the iPS cells cultured in Example 4 and the cell culture system of the present invention, and the phase contrast microscope of the cell adhesion surface after the cells were collected by liquid feeding It is a figure which shows an image.
  • FIG. 27 is an exploded perspective view of the cell culture container in the tenth embodiment of the present invention.
  • FIG. 28A is a perspective view of a cell culture container in the tenth embodiment of the present invention.
  • 28B is a cross-sectional view taken along line AA in FIG. 28A.
  • FIG. 29A is a plan view of a cell culture system in the tenth embodiment of the present invention.
  • FIG. 29B is a partial schematic cross-sectional view taken along line AA in FIG. 29A.
  • FIG. 29C is a diagram showing an example of the operation of the cell culture system in the tenth embodiment of the present invention.
  • FIG. 30A is a plan view of the cell culture system in the eleventh embodiment of the present invention.
  • 30B is a schematic cross-sectional view taken along line AA in FIG. 30A.
  • FIG. 30C is a diagram showing an example of operation of the cell culture system in the eleventh embodiment of the present invention.
  • the cell culture container in the first embodiment of the present invention is a cell culture container for floating cells or adherent cells.
  • Adherent cells such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell
  • a pluripotent stem cell for example, an embryonic stem cell (ES cells) and inducible pluripotent stem cells (iPS cells).
  • Suspension cells include blood cells, preferably T cells and B cells.
  • the cell culture container 10 includes one substrate 15, the other substrate 20 disposed to face the one substrate, and the one substrate.
  • a side wall 16 that forms a culture chamber 17 and a flow path 13 is provided between the other substrate.
  • at least one of the one substrate 15 and the other substrate 20 in contact with the culture chamber 17, for example, the other substrate 20 forms the cell adhesion surface 20 a.
  • One substrate 15 is made of a stretchable material at least in a region corresponding to the planar shape of the culture chamber 17 (that is, a region facing the culture chamber 17).
  • one substrate may be referred to as a first substrate and the other substrate may be referred to as a second substrate.
  • the cell culture vessel 10 may include a plurality of culture chambers 17 in the cell culture vessel 10 as shown in FIGS. 1 to 3, but is not particularly limited, and there may be only one culture chamber 17. Good.
  • each culture chamber 17 may be connected by a flow path 13 as shown in FIG. 1, but is not particularly limited.
  • the inlet 11 and the outlet 12 may be provided independently of each other.
  • the culture chamber 17 may include a plurality of channels 13 connected to the inlet 11 and / or channels 13 connected to the outlet 12.
  • the inlet 11 is an inlet of the solution to be sent
  • the outlet 12 is an outlet of the solution to be sent.
  • the inlet 11 and the outlet 12 do not necessarily need to be provided separately, and may be provided integrally.
  • the culture chamber 17 is connected to the two flow paths 13 installed opposite to each other, the solution is introduced from one flow path 13, and the solution is discharged from the other flow path 13. By doing so, a unidirectional and stable flow can be generated in the solution fed in the culture chamber 17.
  • the culture vessel 10 includes a plurality of culture chambers 17, for example, the culture chamber 17 and the flow channel 13 are arranged in a flow channel arrangement as shown in FIG. Can be arranged. Unidirectional and stable flow is preferable for uniformly seeding the cell suspension in the culture vessel or for uniformly perfusing the cell culture medium. In the channel arrangement shown in FIG.
  • the channel 13 into which the solution is introduced into the culture chamber 17 is It is connected to a flow path 13 through which the solution is discharged from another culture chamber 17 adjacent to the culture chamber 17, and the flow path 13 from which the solution is discharged from the culture chamber 17 is further separated from the culture chamber 17. Is connected to the flow path 13 through which the solution is introduced into the culture chamber 17.
  • the channel 13 into which the solution is introduced into the culture chamber 17 is connected to the inlet 11.
  • the flow path 13 through which the solution is discharged from the culture chamber 17 is connected to the flow path 13 through which the solution is introduced into another culture chamber 17 adjacent to the culture chamber 17.
  • the channel 13 into which the solution is introduced into the culture chamber 17 is adjacent to the culture chamber 17.
  • the flow path 13 from which the solution is discharged from another culture chamber 17 is connected, and the flow path 13 from which the solution is discharged from the culture chamber 17 is connected to the outlet 12.
  • the one substrate 15 of the cell culture container 10 is formed of a stretchable material. Thereby, as will be described later, it is possible to form a concavo-convex shape on one substrate 15 or to eliminate the concavo-convex shape. Further, as will be described later, one substrate 15 can be pushed into the culture chamber 17 by the pressing body.
  • One substrate 15 is preferably transparent from the viewpoint of facilitating microscopic observation, and more preferably colorless and transparent. Examples of such stretchable materials include polydimethylsiloxane (PDMS) and silicone.
  • PDMS polydimethylsiloxane
  • One substrate 15 is preferably formed of silicone rubber. Thereby, flexibility and strength can be imparted to one substrate 15.
  • One substrate 15 is not particularly limited, and has a thickness of, for example, 0.01 mm to 0.3 mm, 0.05 mm to 0.1 mm, or 0.02 mm to 0.2 mm.
  • the hardness of one substrate 15 can be 20 to 65, preferably 20 to 30 in terms of type A durometer hardness.
  • the thickness of one substrate 15 is preferably 0.05 mm to 0.3 mm.
  • the inner surface of the culture chamber 17 other than the cell adhesion surface for example, the inner surface of the culture chamber 17 other than the cell adhesion surface 20a is preferably non-cell-adhesive and is coated with a cell non-adhesion coating for the purpose of preventing cell adhesion. It may be.
  • the cell non-adhesive coating is not particularly limited as long as it is a cell non-adhesive coating, but celluloses such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymer; polyvinyl Polypyrrole; Polyethylene glycol; Polylactic acid; Polyamides such as polyacrylamide and polyN-isopropylacrylamide; Polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, dextran, and cells thereof A coating with a non-adhesive derivative is mentioned. From the viewpoint of high transparency and good visibility, polyethylene glycol is preferred.
  • the cell adhesion surface 20a of the other substrate 20 is preferably cell adhesion, and may be coated with a cell adhesion coating for the purpose of imparting cell adhesion.
  • a cell adhesion coating for the purpose of imparting cell adhesion.
  • a cell-adhesive basement membrane matrix such as Matrigel (trademark) (manufactured by BD Falcon)
  • Matrigel trademark
  • a cell-adhesive coating can be applied to the adhesive surface.
  • the coating can be performed by a method well known to those skilled in the art.
  • a commercially available coating agent it can be coated according to the description in the manufacturer's manual.
  • the thickness and hardness of the other substrate 20 may be the same as or different from those of the one substrate.
  • the other substrate 20 is preferably transparent from the viewpoint of facilitating microscopic observation.
  • the other substrate 20 is not particularly limited, but is preferably a gas permeable membrane from the viewpoint of maintaining the oxygen concentration and carbon dioxide concentration in the culture chamber.
  • it is made of polystyrene for cell culture as a gas permeable membrane for cell culture.
  • Gas permeable membranes are commercially available.
  • the gas permeable membrane plays an important role in supplying oxygen or the like to the culture medium in the culture chamber 17.
  • the use of a gas permeable membrane having flexibility is advantageous in that the cells can be effectively peeled off from the membrane surface by ultrasonic vibration, and the number of options for the cell peeling method is increased.
  • the other substrate 20 may be formed of a non-stretchable material, but may be formed of a stretchable material like the one substrate 15.
  • both the one substrate 15 and the other substrate 20 are made of an elastic material, the volume variation of the culture chamber 17 due to the one substrate 15 forming an uneven shape causes the other substrate 20 to expand and contract. Can be absorbed. Thereby, even if the outlet and the inlet are sealed using a rubber plug or the like and the unevenness is formed on one of the substrates 15, the risk of liquid leakage from the outlet and the inlet can be suppressed.
  • both the one substrate 15 and the other substrate 20 are made of a stretchable material, the volume variation of the culture chamber 17 that occurs when the pressing body is pushed toward the one substrate 15 is caused to be outward on the other substrate 20 side. It can be absorbed by swelling. Therefore, the risk of liquid leakage from the inlet 11 and the outlet 12 that can be sealed with a rubber stopper or the like can be suppressed.
  • the other substrate 20 is formed of a stretchable material
  • the other substrate 20 is preferably formed of silicone rubber. Thereby, flexibility and strength can be imparted to the other substrate 20.
  • the side wall 16 of the cell culture vessel 10 is not particularly limited, but can be made of, for example, plastic, for example, made of polystyrene, polypropylene, polycarbonate, or acrylic.
  • the other substrate 20 can be made of plastic, for example, and can be made of polystyrene, polypropylene, polycarbonate, or acrylic, for example.
  • the side wall 16 of the cell culture vessel 10 can also be formed of silicone rubber. Thereby, flexibility and strength can be imparted to the side wall 16 of the cell culture container 10.
  • the side wall 16 of the cell culture vessel 10 has a wall portion that forms the peripheral wall of the cell culture vessel 10 and a wall portion that partitions the space in the cell culture vessel 10 and forms the culture chamber 17 and the flow path 13.
  • the one substrate 15 of the cell culture vessel 10 is made of a stretchable material, can be formed with various uneven shapes as will be described later, and can be easily eliminated. Thereby, the cell culture container 10 can form an uneven shape only when necessary, and high visibility can be secured by eliminating the uneven shape when observing the cultured cells. By eliminating the uneven shape, it is possible to save the medium, reagent and sample.
  • the cell subculture system in the first embodiment of the present invention includes a cell culture container 10 (see FIGS. 1 to 3) in the first embodiment of the present invention, and cell culture.
  • the enclosure 10 is provided outside the one substrate 15 of the container 10, and includes an enclosure 30 having a concavo-convex shape 32 on an opposing surface facing the one substrate 15.
  • a vacuum source 92 is connected to the opposing surface of the enclosure, and the opposing surface having the concavo-convex shape 32 of the enclosure 30 is evacuated by operating the vacuum source 92.
  • the one substrate 15 can be adsorbed to the opposing surface of the enclosure 30 having the concavo-convex shape 32, and the concavo-convex shape 34 can be formed on the one substrate 15.
  • the vacuum source 92 is preferably connected to the bottom surface 33 of each concave portion on the opposing surface.
  • the concavo-convex shape 34 formed on one substrate includes one substrate 15 and the enclosure 30 having the concavo-convex shape 32 on the opposing surface corresponding to the one substrate, between the one substrate and the enclosure. It is a shape formed by contacting the substrate 15 so as to form a vacuum chamber 35 and evacuating the vacuum chamber 35 so that one substrate 15 is adsorbed to the opposing surface having the concavo-convex shape 32 of the surrounding body 30. The shape corresponds to 32.
  • the concavo-convex shape 34 formed on one substrate can be used as a microwell used to form a cell clump.
  • the cell subculture system in the present embodiment includes a fixture 80 that holds the cell culture container 10 and the enclosure 30 as shown in FIG. 4C.
  • the fixture 80 can fix the cell culture container 10 and the enclosure 30 so as not to move in a state where the fixture 80 is in contact with the substrate 15 so as to form a vacuum chamber 35 between the enclosure 30.
  • a fixture that clamps the cell culture container 10 and the enclosure 30, for example, a clamp can be used as the fixture 80.
  • the fixture 80 can be connected via a rotation shaft 81 to a rotation mechanism 80A that reverses the cell culture container 10 and the enclosure 30 by rotation.
  • the cell subculture system in the present embodiment having the fixture 80 equipped with the rotation mechanism 80A can be preferably used for culturing adhesive cells. Therefore, the cell culture passage system in this embodiment having the fixture 80 provided with the rotation mechanism 80A can be a cell culture passage system for culturing adhesive cells.
  • the inner surface of one substrate 15 preferably has cell non-adhesiveness and the inner surface of the other substrate 20 has cell adhesiveness as a cell culture container. Use cell culture vessels.
  • the cell subculture system in the present embodiment may include a valve 90 that maintains the vacuum state by closing the vacuum line 91 after the vacuum chamber 35 is evacuated.
  • a valve 90 that maintains the vacuum state by closing the vacuum line 91 after the vacuum chamber 35 is evacuated.
  • the uneven shape 34 on the one substrate 15 can be maintained even after the vacuuming by the vacuum source 92 is stopped.
  • the cell culture container 10 and the enclosure 30 are separated from the vacuum line 91 connected to the vacuum source 92 through the valve 90 portion, and the cell culture container 10, the enclosure 30 and the valve 90 are transported to another place.
  • the vacuum line 91 and the valve 90 may be detachably attached to the enclosure 30 or may be integrated with the enclosure 30.
  • a vacuum refers to the state decompressed from atmospheric pressure.
  • microwell examples include the shape of a microwell carved on the bottom surface of a commercially available AggreWell (trademark) (manufactured by STEM CELL TECHNOLOGIES) (that is, a quadrangular pyramid shape).
  • AggreWell commercially available AggreWell (trademark) (manufactured by STEM CELL TECHNOLOGIES) (that is, a quadrangular pyramid shape).
  • cell clumps can be formed using the following principle. First, when cells are seeded in microwells, the cells naturally settle by gravity in the cell culture medium. Therefore, when cells are seeded in a microwell (indent) having an inclination, the cells gather in the microwell by utilizing the inclination of the well. The cells then form cell clumps by forming cell adhesions with neighboring cells.
  • the microwell has a shape that collects when the cells sink, for example, a shape that becomes deeper as the inner circumference approaches the bottom surface. That is, the microwell preferably has, for example, a cone shape, a round bottom, a V bottom, and a U bottom.
  • the shape of the opening of each well that opens toward the culture chamber can be appropriately selected in consideration of the ease of processing and the shape in which a large number of wells can be arranged. For example, a triangle, a square, or a hexagon Can have a polygonal shape or a circular shape.
  • a well for forming a cell clump is referred to as a “microwell” in order to distinguish it from a well for partitioning a culture chamber of a multiplate used for normal cell culture.
  • Microwell is not a term intended to exclude wells having openings with a side or diameter of 1 mm or more, and “microwell” includes wells with openings with a side or diameter of 1 mm or more. In the present specification, the microwell is sometimes referred to as a “dent”.
  • the size of the opening of the microwell can be determined by the size of the cell clump to be formed, and has an area equivalent to, for example, a circle having a diameter of 100 ⁇ m to 3 mm, a diameter of 200 ⁇ m to 800 ⁇ m, or a diameter of 400 ⁇ m to 600 ⁇ m. An opening can be provided.
  • the cell agglomerates to be formed have a certain size. From the viewpoint of uniforming the size of the cell clumps to be formed, it is preferable that the shape of the microwell formed on one substrate of the cell culture container is uniform. By doing so, it becomes easy to disperse the cells in the microwells relatively uniformly, and as a result, it becomes possible to form a cell aggregate having a uniform size.
  • microwells are arranged on the bottom surface of the container, and the microwells have no gaps (so that there is no flat portion between adjacent wells). ) Or the gap between the microwells is preferably arranged to be minimized.
  • the microwells are aligned at equal intervals on the plane.
  • the concavo-convex shape 32 of the enclosure 30 has the shape and arrangement of the recesses 34 formed by being adsorbed to the concavo-convex shape 32, and the shape of the microwell. It is preferable that it is formed so as to be arranged.
  • the enclosure is detachable, and the enclosure can be replaced with a desired enclosure.
  • corrugated shapes 34 are formed in one board
  • FIG. be able to. For example, if you want to obtain a large cell clump, you can use an enclosure with a large concave and convex shape, and if you want to obtain a small cell clump, each concave is small. An enclosure can be used.
  • the cell subculture system in the first embodiment may further include a cell culture container rotation mechanism 80 ⁇ / b> A that rotates the cell culture container 10 and the enclosure 30 to invert the top and bottom.
  • the cell subculture system in the first embodiment further includes a liquid feeding means for feeding a solution such as a cell suspension, a cell culture medium, a cell detachment solution, and a cell recovery solution to the channel and the culture chamber. Also good.
  • the cell subculture system in the first embodiment may further include temperature control means for controlling the temperature of the culture chamber or the temperature around the culture chamber.
  • the cell subculture system in the first embodiment may further include humidity control means for controlling the humidity around the culture chamber.
  • the cell culture container may be a cell culture container that is removable from other parts of the cell subculture system.
  • the cell subculture system in the first embodiment may include an incubator including an atmosphere control unit capable of controlling the internal temperature and humidity to a temperature and humidity suitable for cell culture.
  • an uneven shape is formed on one substrate of the cell culture container in the first embodiment using the cell subculture system in the first embodiment.
  • a method for forming the film will be described.
  • adherent cells are seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20a formed by the other substrate 20. it can.
  • FIG. 4A is a plan view of the enclosure 30 used in the cell culture system in the first embodiment
  • FIG. 4B is a side cross-sectional view of the AA cross section
  • FIG. 4C is a side cross-sectional view taken along the line AA shown in FIG. 4A in a state in which the cell culture container and the enclosure 30 in the first embodiment are in contact with each other in the cell culture system in the first embodiment. is there.
  • FIG. 4D shows the cell passage in a state in which one substrate 15 is adsorbed to the uneven shape 32 of the enclosure 30 and the uneven shape 34 is formed on the one substrate 15 in the cell culture system in the first embodiment.
  • Fig. 4E is an upper plan view of the culture system
  • Fig. 4E is a side sectional view of the AA section.
  • the culture chamber 17 and the flow path 13 of the cell culture container 10 are filled with the cell culture medium.
  • the enclosure 30 is brought into contact so that the vacuum chamber 35 is formed outside the one substrate 15 of the cell culture container 10 in the first embodiment.
  • the vacuum source 92 connected to each concave-part bottom face 33 of the enclosure 30 is operated, and the opposing surface having the uneven shape of the enclosure 30 is evacuated.
  • the uneven shape 34 is formed on one substrate 15.
  • the cell subculture system in the first embodiment includes the valve 90 that maintains the vacuum state after the vacuum chamber 35 is evacuated, if the uneven shape 34 is formed on one substrate 15, the valve 90 can be closed. Then, even if the evacuation of the vacuum chamber 35 is stopped, the uneven shape 34 can be maintained.
  • the cell culture container 10 and the enclosure 30 are separated from the vacuum line 91 connected to the vacuum source 92 through the valve 90 portion, and the cell culture container 10, the enclosure 30 and the valve 90 are separated from each other (for example, an incubator). May be conveyed.
  • the concave / convex shape 34 is formed and eliminated on one substrate 15
  • the volume of the culture chamber 17 varies with the formation of the concave / convex shape, and the required medium amount changes.
  • the amount of the medium in the culture chamber 17 may be adjusted by feeding the medium to 17 or discharging the medium from the culture chamber 17. For example, when a negative pressure is applied to one of the substrates 15, the required amount of medium increases with the formation of the uneven shape. Therefore, when the uneven shape is formed, the medium may be fed to the culture chamber 17 to compensate for the increase in the necessary medium amount.
  • one substrate 15 is made of a stretchable material, and the uneven shape 34 formed on one substrate 15 is easily eliminated by releasing the vacuum.
  • the negative pressure on one substrate 15 is released, the required amount of medium decreases as the uneven shape is eliminated. Therefore, when the uneven shape is eliminated, the medium may be discharged from the culture chamber 17 to adjust the amount of decrease in the necessary medium amount.
  • the cell subculture system in the first embodiment provided with the cell culture container 10 and the enclosure 30 in the first embodiment has the uneven shape 34 formed on one substrate 15 and formed.
  • the uneven shape 34 can be eliminated.
  • the uneven shape 34 can be formed on one substrate 15 of the cell culture container. And the uneven
  • the irregular shape is made into a certain shape and cells are seeded in the recess (microwell) formed by this irregular shape, the cells form a cell agglomeration of a certain size.
  • each cell clump exhibits the same growth rate, and the passage timing of each colony can be synchronized. Therefore, this method is very advantageous in establishing a fully automated cell subculture system.
  • FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, and FIG. 8E explain a method for subculturing cells when the cell subculture system in the first embodiment is used.
  • 8A to 8E show a cell subculture system in a second embodiment to be described later, but only the principle of forming an uneven shape on one substrate 15 is different, and cells are subcultured. The principle and process are not different from the method of subculturing cells in the first embodiment.
  • FIGS. 8A to 8E are diagrams showing a method of forming a cell clump on one substrate 15 of the cell culture container in the second embodiment and seeding the cell clump on the other substrate 20.
  • FIG. is there.
  • the cell culture container in the second embodiment is filled with a cell suspension.
  • the vacuum chamber 64 is evacuated to form a large number of microwells on one substrate of the cell culture container.
  • the cells in the cell suspension fall into the formed microwells to form cell clumps.
  • the formation of the microwell can be performed while further supplying a culture medium or a cell suspension to the culture chamber 17 in order to cope with an increase in the volume of the culture chamber 17.
  • FIG. 8C the rotation mechanism 80A is driven to rotate the fixture 80, so that the cell culture container 10 and the enclosure 60 of the present invention are turned upside down, and the formed cell conglomerate is transferred to the other side. Sowing on the substrate 20. If some cell clumps still adhere to one substrate 15 and have not been seeded on the other substrate 20 (eg, FIG. 8C), then the vacuum in the vacuum chamber 64 is further removed, as shown in FIG. 8D. The pulling is released to eliminate the uneven shape of one substrate 15, thereby dropping the cell clump attached to one substrate 15 onto the other substrate 20.
  • FIG. 8E is a diagram showing a state in which cells are then cultured and each cell clump forms a colony. In FIGS. 8B-8E, the culture chamber 17 is filled with the medium. Note that the steps of FIGS. 8C to 8D may be repeated.
  • the cell passage method using the cell passage culture system in the first embodiment is: (A) a step of dispersing cells during passage; (B) injecting the dispersed cells into the culture chamber 17 of the cell culture vessel 10; (C) forming a concavo-convex shape 34 on one substrate 15 facing the culture chamber 17 of the cell culture container; (D) forming a cell clump in the cells seeded in the concave / convex recesses 34 formed on one substrate 15. Either of the steps (B) and (C) may be performed first.
  • the cell passage method using the cell passage culture system in the first embodiment is: (A) a step of dispersing cells during passage; (B) a step of injecting the cells dispersed in step (A) into the culture chamber 17 of the cell culture vessel 10; (C) forming a concavo-convex shape on one substrate 15 facing the culture chamber 17 of the cell culture container, and seeding cells in the concavo-convex recess 34; (D) and a step of forming a cell clump in the cells seeded in the concave-convex recess 34 formed on one substrate 15.
  • the cell suspension may be injected into the culture chamber 17 after the concave and convex shape is formed on one substrate 15.
  • the cell passage method using the cell passage culture system in the first embodiment is: (A) a step of dispersing cells during passage; (B ′) forming a concavo-convex shape on one substrate 15 facing the culture chamber 17 of the cell culture container; (C ′) a step of injecting the cells dispersed in the step (A) into the culture chamber 17 of the cell culture vessel 10 and seeding in the concave / convex shaped recesses 34 formed on the one substrate 15; (D) It is good also as a thing including the process of forming a cell clump in the cell seed
  • the cell passage method of the present invention further includes, after steps (A) to (D), (E) a step of inverting the cell culture vessel and seeding the formed cell clump on the other substrate. You may go out.
  • the cell passage method of the present invention may further include a step of culturing after step (E), that is, (E ′) culturing the seeded cells on the other substrate 20, or After the steps (A) to (D), the method may further include a step (F) of culturing the cell clump formed in the step (D) on the one substrate 15, so that the cells are passaged. And culturing.
  • the cell passage method of the present invention may further include a step (G) of recovering the cultured cells.
  • pluripotent stem cells that are physiologically or physically detached from the cell adhesion surface can be used in the adhesion culture.
  • an enzyme for detaching pluripotent stem cells from the cell adhesion surface an enzyme used in a conventional method can be used, and for example, an enzyme such as trypsin, dispase, actase or collagenase can be used.
  • the detachment of pluripotent stem cells from the cell adhesion surface may also be performed using a chemical substance having a cell detaching action, such as a chelating agent of divalent ions (particularly Mg 2+ ) such as ethylenediaminetetraacetic acid (EDTA), These may be performed in combination with the above enzyme.
  • a chemical substance having a cell detaching action such as a chelating agent of divalent ions (particularly Mg 2+ ) such as ethylenediaminetetraacetic acid (EDTA), These may be performed in combination with the above enzyme.
  • vibration such as high-frequency vibration may be applied to the cell adhesion surface, and / or a cell scraper may be used.
  • the cells may be detached by combining the above physiological detachment method and physical detachment method. Those skilled in the art will be able to appropriately remove the pluripotent stem cells from the cell adhesion surface using the well-known peeling method as described above.
  • the cell mass can be dissociated to the single cell level.
  • the dissociation of the cell mass may be performed simultaneously with the detachment of the cell from the culture surface, or may be performed after the cell mass is detached.
  • the exfoliated cell mass can be dispersed after being dissociated to a single cell level by, for example, pipetting water flow.
  • “dispersing to a single cell level” means a cell mass such that the average number of cells contained in one cell mass is 1 to 100, preferably 1 to 10. Is dissociated and then dispersed, and includes completely dissociating into single cells and then dispersing. Therefore, in the present invention, the cell mass may be dissociated after being completely dissociated into single cells, or may be dispersed after being dissociated so that most of the cells become single cells.
  • cell mass after dispersion is large, when the cell mass is seeded in the microwell, the number of cells seeded in each microwell tends to vary, and thus the cell mass after dispersion is preferably small.
  • the cell mass separated from the culture surface may be dispersed to the single cell level by further processing using an enzyme.
  • Enzymes that can be used to dissociate cells to the single cell level include those that can break cell-cell bonds and cell-extracellular matrix (ECM) bonds. Can be mentioned and these enzymes are well known to those skilled in the art. Dissociation of the cell mass using an enzyme or a water flow can be automated, and the process (A) can be automated.
  • a compound that suppresses adverse effects eg, cell death
  • Y-27632 and other ROCK inhibitors can be added.
  • Step (B) Step of injecting the cells dispersed in the step (A) into the culture chamber 17 of the cell culture container 10
  • the suspension of the cells dispersed in the step (A) is introduced into the inlet 11 of the cell culture container 10.
  • the cell suspension is injected into the cell culture vessel by an injection device (not shown) connected to the inlet 11.
  • step (C) Cells or cells obtained by dispersing in the step (A) in which a concave-convex shape is formed on one substrate 15 facing the culture chamber 17 of the cell culture container and the cells are seeded in the concave-convex recess 34
  • a clump (hereinafter sometimes simply referred to as a “cell”) can be successfully grown after passage after forming a cell clump. Formation of a cell clump can be performed by forming the concavo-convex shape (microwell) on one substrate 15 and seeding the obtained cells in the dent.
  • the cells obtained by dispersing in step (A) naturally sink due to gravity in the culture solution.
  • the cells when cells are seeded in a well (indent) having a slope, the cells gather in the well by utilizing the slope of the well. Thereafter, the cells adhere to neighboring cells to form a cell clump. If the concavo-convex shape is formed on one substrate 15 after the cell suspension is injected in the step (b), the cells in the suspension fall into the concavo-convex shape and gather at the bottom of the well.
  • the cell agglomerates to be prepared have a uniform size.
  • the size of the cell conglomerate to be produced is determined by the number of cells to be seeded in each concave and convex shape, in step (c), the number of cells to be seeded in each concave is made uniform. It is preferable.
  • the fixed amount means that the average number of cells seeded in each microwell in step (c) is, for example, 10 to 3,500 (that is, the average diameter of the formed cell clump is 35 to 350 ⁇ m).
  • the average diameter of the formed cell clumps is from 50 to 200 ⁇ m), more preferably from 40 to 500 (ie the average of the formed cell clumps) It means that the diameter is 60 to 160 ⁇ m), more preferably 55 to 220 (that is, the average diameter of the formed cell clumps is 69 to 115 ⁇ m).
  • the cells are sufficiently suspended in the suspension. What is necessary is just to liquid-feed to a culture room. The sent cells can spontaneously fall into the microwell by gravity.
  • the cell subculture system in the first embodiment includes the fixture 80, one substrate of the cell culture container is brought into contact with the enclosure 60 having an uneven shape on the surface facing the one substrate. After that, the cell culture container 10 and the enclosure 60 are fixed by the fixing tool 80.
  • the cell subculture system in the first embodiment includes a valve that maintains the vacuum state of the vacuum chamber 35, when the uneven shape 34 is formed on one substrate 15, the valve 90 is closed to perform vacuum.
  • the evacuation is stopped by closing the vacuum line (gas flow path) 91 connecting the chamber 35 and the vacuum source 92, the uneven shape 34 can be maintained even after the evacuation is stopped.
  • the culture chamber 17 undergoes a volume change.
  • the concave-convex shape 34 can be formed on one substrate 15 while feeding the culture chamber 17.
  • the surface of one substrate 15 is non-adhesive to the cells.
  • a coating can be applied.
  • the number of cells seeded in each dent can be adjusted as appropriate.
  • seeding of the cells in the microwell can be performed uniformly by sufficiently suspending the cells. Since these operations can be automated, the step of seeding a certain amount of cells in a microwell in step (c) can also be fully automated.
  • Step (B ′) Step of forming an uneven shape on one substrate 15 facing the culture chamber 17 of the cell culture container
  • the cell culture container in the first embodiment forms an uneven shape on one substrate 15 as described above.
  • a vacuum chamber is formed between one substrate and the enclosure by bringing one substrate of the cell culture container into contact with an enclosure having a concavo-convex shape on the surface facing the one substrate. Then, the vacuum chamber is evacuated and one substrate is adsorbed to the uneven shape of the surrounding body to form the uneven shape on the one substrate.
  • Step (b ′) can be fully automated.
  • the cells dispersed in the step (A) are injected into the culture chamber 17 of the cell culture vessel 10 and seeded in the concave and convex shape formed in the step (b ′), and dispersed in the step (A).
  • Cells or cell masses obtained in this manner (hereinafter sometimes simply referred to as “cells”) can be proliferated well after passage after forming a cell clump.
  • Formation of a cell clump can be performed by seeding the obtained cells in a microwell on one substrate 15.
  • the seeding of the cells in the microwells on one substrate 15 can be performed by feeding the cell suspension into the culture chamber 17 with one substrate 15 facing down.
  • the cells obtained by dispersing in step (A) naturally sink due to gravity in the culture solution. Accordingly, when cells are seeded in a well (indent) having a slope, the cells gather in the well by utilizing the slope of the well. Thereafter, the cells adhere to neighboring cells to form a cell clump.
  • the cell agglomerates to be prepared have a uniform size.
  • the size of the cell conglomerate to be produced is determined by the number of cells seeded in each concavo-convex recess, in step (c ′), the number of cells seeded in each recess is uniform.
  • the fixed amount means that the average number of cells seeded in each microwell in the step (c ′) is, for example, 10 to 3,500 (that is, the average diameter of the formed cell cluster is 35 to 35).
  • step (c ′) In order to make the number of cells seeded in each microwell constant in step (c ′), after adjusting the concentration of the cells in the cell suspension, the cells are sufficiently suspended in the suspension. To the culture chamber. The sent cells can spontaneously fall into the microwell by gravity.
  • the cell subculture system in the first embodiment includes the fixture 80, one substrate of the cell culture container is brought into contact with the enclosure 60 having an uneven shape on the surface facing the one substrate. After that, the cell culture container 10 and the enclosure 60 are fixed by the fixing tool 80.
  • the cell subculture system in the first embodiment includes a valve that maintains the vacuum state of the vacuum chamber 35, when the uneven shape 34 is formed on one substrate 15, the valve 90 is closed to perform vacuum.
  • the evacuation is stopped by closing the vacuum line (gas flow path) 91 connecting the chamber 35 and the vacuum source 92, the uneven shape 34 can be maintained even after the evacuation is stopped.
  • the surface of one substrate 15 is non-adhesive to the cells.
  • a coating can be applied.
  • the number of cells seeded in each dent can be adjusted as appropriate.
  • seeding of the cells in the microwell can be performed uniformly by sufficiently suspending the cells. Since these operations can be automated, the step of seeding a certain amount of cells in a microwell in step (c ′) can also be fully automated.
  • (D) Step of forming cell clumps in cells seeded in concave and convex dents formed on one substrate As shown in FIG. 8B, cells seeded in concave and convex dents formed on one substrate When left undisturbed, they gather at the bottom of each dent by gravity and the cells adhere to form a cell clump. The cells may be collected at the bottom of the recess by centrifuging the cell culture container of the present invention using a centrifugation technique.
  • centrifugation at 400 g to 3000 g for 1 minute to 10 minutes can be performed. By doing in this way, a cell can be effectively collected on the bottom face of a dent.
  • the centrifugation technique is not necessarily used. There is no need to use. That is, after seeding cells that have been dispersed to the single cell level in the dent and then using a method of centrifugation, for example, the cells can be collected in pluripotent stem cells within a few hours just by leaving the cells in the dent. A lump can be formed. Thus, in the first embodiment, cell clumps can be formed without using a centrifugation technique.
  • cell clumps can be formed in pluripotent stem cells by standing in a dent.
  • the standing time may be longer than the time necessary for the pluripotent stem cells to form a cell clump, for example, 8 hours or longer.
  • the standing time can be, for example, 8 hours to 24 hours, preferably 8 hours to 16 hours, and more preferably 8 hours to 12 hours.
  • the body 60 may be separated from the vacuum line 91 connected to the vacuum source 92 through the valve 90 portion, and the cell culture vessel 10, the enclosure 60, and the valve 90 may be transported to the incubator and left in the incubator.
  • the shorter the standing time the looser the connection between the cells in the cell clump, so that the cell clump is more fragile, while the cell clump develops quickly after seeding in the culture vessel. There is.
  • the cell agglomerates prepared in the step (D) are arranged in a certain size.
  • the average number of cells contained in each cell clump is, for example, 10 to 3,500 (that is, the average diameter of the cell clump is 35 to 350 ⁇ m), preferably 25 to 870. (That is, the average diameter of the cell clump is 50 to 200 ⁇ m), more preferably 40 to 500 (that is, the average diameter of the cell clump is 60 to 160 ⁇ m), and more preferably 55 to 220.
  • the average diameter of the cell clumps is 69 to 115 ⁇ m
  • the average diameter of the cell clumps can be appropriately adjusted according to the size of the dent and the number of cells to be seeded.
  • the dent can be selected to be larger than the size of the cell clump to be produced.
  • step (E) Step of inverting the cell culture container upside down and seeding the formed cell agglomeration on the other substrate
  • the irregularities formed on the one substrate 15 in step (D) in the cell culture container in the first embodiment The cell agglomerate formed in the shape recess can then be seeded on the other substrate 20 of the cell culture vessel.
  • the step (E) can be preferably performed, for example, when culturing adhesive cells, and the cell adhesion surface 20a of the other substrate 20 can be made cell adhesive.
  • step (E) cell seeding can be precisely seeded.
  • the rotation mechanism provided in the fixing tool 80 is driven to turn the cell culture container 10 upside down so that the cell agglomeration is transferred to the other side.
  • the cell agglomeration can be precisely seeded on the culture surface (for example, the other substrate 20) of the cell culture container.
  • the cell agglomerates fall almost vertically from the dents onto the culture surface of the culture container. Will be sown on top.
  • the dent can be designed based on the arrangement of cell clumps after dropping on the culture surface.
  • the cell clumps can be uniformly seeded on the culture surface of the culture container, and the cells can be uniformly propagated on the culture surface.
  • the seeded cell cluster can be rapidly developed after seeding, and then cultured well in a state where pluripotency is maintained. From the viewpoint of uniformly seeding the cell clumps on the culture surface of the culture vessel, it is preferable to seed the cell suspension containing the cell clumps after sufficient suspension.
  • the comparison Cell clumps can be aligned on the culture surface of the other substrate.
  • the negative pressure applied to one substrate 15 is released and formed on one substrate. It may be dissociated by eliminating the uneven shape thus formed and thereby reducing the area of the contact surface between the cell agglomerate and one substrate 15.
  • the cell clump when the cell clump is still adhered to the surface of the dent after the upside down, the cell clump can be dissociated from one substrate by various methods. .
  • FIG. 9A is a diagram illustrating a state corresponding to FIG. 8D.
  • FIG. 9A even after the uneven shape of one substrate 15 is eliminated, some cell clumps still adhere to one substrate 15.
  • FIG. 9B is a diagram for explaining a method of supplying gas to the vacuum chamber and pushing one substrate 15 into the culture chamber 17, thereby dissociating the cell clump from one substrate 15 and dropping it onto the other substrate 20.
  • FIG. 9C is a diagram for explaining a method of repeatedly evacuating the vacuum chamber and supplying the gas to the vacuum chamber, thereby dissociating the cell clump from one substrate 15 and dropping it onto the other substrate 20.
  • the culture chamber 17 is filled with the medium.
  • 9A, 9B, and 9C the cell culture container 10 and the enclosure 60 are fixed by a fixture 80.
  • the adhesion between one substrate and the cell aggregate is performed by supplying a gas to the vacuum chamber and pushing the one substrate 15 into the culture chamber 17 to contact the cell aggregate with the one substrate 15. It may be dissociated by reducing the surface area, or may be dissociated by repeating evacuation of the vacuum chamber and supply of gas to the vacuum chamber, as shown in FIG. 9C.
  • step (E ′) a step of culturing cells on the other substrate 20 may be further performed. Further, in the step (E), from the viewpoint of facilitating the microscopic observation of the cultured cells, a step of eliminating the uneven shape formed by applying a negative pressure to the one substrate 15 from the outside may be further performed. Good. When performing the process of eliminating the uneven shape, the volume of the culture chamber 17 changes, so the amount of required medium changes. Step (E) When performing the step of eliminating the concavo-convex shape, the medium is preferably fed to the culture chamber 17 or discharged from the culture chamber 17 to adjust the amount of the culture medium in the culture chamber 17 to an appropriate amount. be able to.
  • the state of cell agglomeration from one substrate 15 can be monitored using, for example, a CCD camera (not shown).
  • step (F) In the step (F) of culturing the cell clump formed by seeding in the dent on one substrate , the cells are cultured without inverting the cell culture container in the first embodiment ( For example, FIG. 10A to FIG. 10C).
  • one substrate 15 is coated with a cell non-adhesive coating, for example, and can be preferably performed by cell culture of floating cells, for example.
  • FIG. 10A is a diagram showing a state in which the cell culture container in the first embodiment is filled with the cell suspension.
  • the vacuum chamber 64 is evacuated to form a large number of microwells on one substrate of the cell culture container in the first embodiment, and cells are seeded in the formed microwells. To form cell clumps.
  • FIG. 10C without performing the top-and-bottom inversion operation of the culture vessel, the vacuuming of the vacuum chamber is released to eliminate the uneven shape formed on one substrate, and the one substrate 15 is left as it is. Cells are cultured on the cell adhesion surface 15a.
  • the culture chamber 17 is filled with the medium.
  • step (F) a step of culturing cells may be further performed.
  • a step of eliminating the uneven shape formed by applying a negative pressure to the one substrate 15 from the outside may be further performed. Good.
  • the volume of the culture chamber 17 changes, so the amount of required medium changes.
  • Step (F) When performing the step of eliminating the uneven shape, preferably, the medium is fed to the culture chamber 17 or discharged from the culture chamber 17 to adjust the amount of the culture medium in the culture chamber 17 to an appropriate amount. be able to.
  • Step of collecting cultured cells For example, cells adhered to one substrate 15 or the other substrate 20 of the cell culture container in the first embodiment send a cell detachment solution to the flow path 13 and the culture chamber 17, for example. It can be peeled off from one substrate 15 or the other substrate 20 and collected by applying high frequency vibration to one substrate 15 or the other substrate 20 to which cells are adhered.
  • the uneven shape of one substrate 15 is eliminated in the step (E) or the step (F)
  • the amount of the cell detachment solution fed can be reduced.
  • cell detachment solutions are known as cell detachment solutions, and those skilled in the art can select and use an appropriate cell detachment solution.
  • the cell detachment solution can be a cell culture medium or physiological saline containing trypsin and / or a divalent metal ion chelator.
  • the cell detachment solution can be a cell culture medium or physiological saline containing trypsin and / or a divalent metal ion chelator.
  • a cell culture medium what does not contain serum can be used.
  • the high-frequency vibration applying means can use ultrasonic waves as the high-frequency vibrations, and the ultrasonic waves are generated using, for example, commercially available ultrasonic generators for cell disruption and ultrasonic generators for ultrasonic cleaners as high-frequency vibration sources. Can be generated.
  • the high frequency vibration generating source is brought close to or in contact with one substrate 15 or the other substrate 20 from the outside of the closed culture vessel, the surface contacting the culture chamber 17 of the one substrate 15 or the other substrate 20 is used. Cells can be peeled off. The cells detached by applying high-frequency vibration to the cell adhesion surface hardly show physical damage, and the detached cells can be cultured well thereafter.
  • High frequency vibration could also be used to weaken the adhesion between cells and cell adhesion surfaces. That is, high-frequency vibration can be applied simultaneously with the feeding of the cell detachment solution.
  • cells can be subcultured using the cell culture container in the first embodiment.
  • a cell can be subcultured using the cell subculture system in 1st embodiment.
  • Cell culture vessel The cell culture container in the second embodiment is the same as the cell culture container 10 in the first embodiment.
  • Cell subculture system Cell subculture system
  • FIGS. 7A to 7D Cell subculture system
  • the cell subculture system in the second embodiment is different only in the structure of the enclosure, and other configurations are the same as those of the cell subculture system in the first embodiment shown in FIGS. 4A to 4E. .
  • the cell subculture system in the second embodiment is provided on the outer side of the cell culture container 10 and one substrate of the cell culture container, and a plurality of cells are provided on the opposing surface facing the one substrate. And an enclosure 60 having protrusions.
  • a vacuum source 92 is connected to the opposing surface of the enclosure, and the vacuum source 92 is operated to evacuate the opposing surface having a plurality of protrusions of the enclosure. An uneven shape can be formed on one substrate by protrusions.
  • FIG. 7A shows an upper plan view of the enclosure 60
  • FIG. 7B shows a side sectional view of the AA section
  • FIG. 7C also shows that one substrate 15 and the enclosure 60 are brought into contact with each other so as to form a vacuum chamber 64 between the one substrate 15 and the enclosure 60, and then the vacuum source 92 is operated.
  • An upper plan view of the cell subculture system in a state in which the projections and depressions are formed on one substrate by protrusions is shown
  • FIG. 7D is a side sectional view of the AA section.
  • the enclosure 60 faces one substrate 15 when it is brought into contact with one substrate 15 of the cell culture vessel 10 to form a vacuum chamber 64.
  • a plurality of protrusions 61 are provided on the opposing surface.
  • the vacuum chamber 64 is connected to the vacuum source 92 via the connection port 62 with the vacuum source 92, and the vacuum chamber 92 is operated to evacuate the vacuum chamber 64, so that one substrate 15 is recessed by the protrusion 61. 65 can be formed (FIGS. 7C and 7D).
  • the protrusions 61 do not necessarily have to all have the same height, but preferably, as shown in FIGS. 7B and 7D, all the protrusions 61 have the same height, more preferably with the side wall 63 of the enclosure. Accordingly, when one substrate 15 and the enclosure 60 are brought into contact with each other, the protrusion 61 comes into contact with one substrate 15 or the tips of the plurality of protrusions 61 in the vicinity of the one substrate 15. The part is located.
  • the shape of the protrusion 61 is not particularly limited as long as the recess 65 is formed in one substrate 15 when the vacuum chamber 64 is evacuated.
  • the shape of the protrusion 61 can be, for example, a cone shape (for example, a cone) or a columnar shape (for example, a cylinder) from the viewpoint of ease of molding and reduction of a necessary amount of material.
  • the tip of the protrusion 16 has a round shape of R0.3 mm to 0.7 mm or R0.4 mm to 0.6 mm. Can do.
  • the arrangement of the protrusions 61 can be appropriately set by those skilled in the art according to the planar arrangement of the recesses 65 to be formed.
  • the protrusions 61 can be aligned in a grid pattern to form a recess 65 aligned in a grid pattern (eg, FIGS. 7A to 7D).
  • the protrusions 61 are arranged at the apexes of parallel hexagons (for example, regular hexagons) in which the plane is filled in a honeycomb shape, so that the recesses 65 aligned in the honeycomb shape can be formed on one substrate 15. it can.
  • the arrangement interval of the protrusions 61 can be, for example, 0.4 mm to 5 mm, for example, 0.8 mm to 3 mm, or for example, 1 mm to 2 mm.
  • cells are seeded in the recess 65 formed on one substrate 15, and the pluripotent stem cells have a cell clump having a diameter of about 50 ⁇ m to 350 ⁇ m (about 25 to about 3 cells). , 500) can be formed.
  • the enclosure 60 may have one or more connection ports 62 with the vacuum source 92.
  • a sealing material such as an O-ring can be interposed at the joint surface between the enclosure 60 and the cell culture vessel 10 of the present invention (for example, the sealing material of FIG. 8B). 21).
  • the enclosure 60 is brought into contact so that the vacuum chamber 64 is formed outside the one substrate 15 of the cell culture container 10.
  • a connection port 62 with a vacuum source 92 is provided on the surface of the enclosure 60 facing the one substrate, the vacuum source 92 is operated, and the vacuum chamber 64 is evacuated.
  • an uneven shape 65 is formed on one substrate 15.
  • one substrate 15 is made of a stretchable material, and the uneven shape 65 formed on the one substrate 15 is easily eliminated by releasing the vacuum.
  • the cell culture system according to the second embodiment including the cell culture container 10 and the enclosure 60 can form an uneven shape on one substrate 15 and eliminate the formed uneven shape.
  • the cell subculture method in the second embodiment is different only in the principle of forming a concavo-convex shape on one substrate of the cell culture container in the step (B), and other parts are the same as those in the first embodiment. It is the same.
  • step (B) the enclosure 60 provided in the subculture system in the second embodiment is used on one substrate of the cell culture container. An uneven shape is formed.
  • FIGS. 5A, 5B, 5E, and 5F Cell culture vessel
  • the cell culture container in the third embodiment shown in FIGS. 5A, 5B, 5E, and 5F is only different in the shape of one substrate 15, and the other configuration is the first configuration shown in FIGS. It is the same as the cell culture container in the embodiment.
  • FIG. 5A, FIG. 5B, FIG. 5E and FIG. 5F the same parts as those in the cell culture system in the first embodiment shown in FIG.
  • FIG. 5A shows an upper plan view of one substrate 15A of the cell culture container 40 in the third embodiment
  • FIG. 5B shows a side sectional view of the AA section
  • 5E shows that in the cell culture container 40 according to the third embodiment, a negative pressure can be applied to the one substrate 15A from the outside of the one substrate 15A to form an uneven shape on the one substrate 15A
  • FIG. 5F is a side cross-sectional view of the cell culture container 40 according to the third embodiment, taken along a cross section corresponding to the AA cross section of FIG.
  • one substrate 15A includes a hard frame portion 42 that forms a plurality of openings, and a stretchable portion 41 that extends into each opening of the frame.
  • the stretchable part 41 in the frame of one substrate extends to form a concave 43 (microwell) uneven shape on one substrate 15A (for example, FIG. 5E).
  • the hard frame part 42 and the expansion / contraction part 41 can be designed according to the desired shape and arrangement of the recesses 43, and when the hard frame part 42 and the expansion / contraction part 41 are arranged in a grid pattern as shown in FIGS. 5A and 5B.
  • the dent is formed in a grid shape.
  • the stretchable part 41 is made of a stretchable material, and the stretchable part 41 can be pulled out of the cell culture container 40 by a constant negative pressure.
  • the hard frame portion 42 may be the same as or different from the stretchable portion 41 regardless of the material, as long as the hard frame portion 42 has a hardness that does not substantially change its shape due to a certain negative pressure.
  • the hard frame portion 42 is made of the same stretchable material as the stretchable portion 41, but has a thickness that does not substantially cause a shape change due to a certain negative pressure.
  • the one substrate 15 ⁇ / b> A can form a recess 43 in each of the stretchable portions 41.
  • the uneven processing of the one substrate 15A is performed outward of the cell culture vessel 40 (FIG. 5E).
  • the hardness of the hard frame portion and the stretchable portion can be appropriately set in relation to the negative pressure applied to the one substrate 15.
  • the hardness of the stretchable portion is the type A durometer hardness. 20 to 65, preferably 20 to 30 (unit).
  • the concave / convex shape is eliminated by forming a concave / convex shape on one substrate 15A and releasing the negative pressure only by applying a negative pressure from the outside to the one substrate 15A. This is advantageous over the cell culture vessel in the first embodiment that requires a complex-shaped enclosure.
  • Cell subculture system Cell subculture system
  • FIGS. 5A, 5B, 5E, and 5F The cell subculture system in the third embodiment is different only in the structure of the cell culture container and the enclosure, and the other configuration is the same as that of the cell subculture system in the first embodiment shown in FIG. is there.
  • the cell subculture system in the third embodiment forms a vacuum chamber between the cell culture container in the third embodiment and one substrate outside the cell culture container. And an enclosure 45.
  • a vacuum source 92 is connected to the vacuum chamber 44. By operating the vacuum source 92 and evacuating the vacuum chamber, the uneven shape 43 is formed on one substrate. It is formed.
  • the enclosure 45 forms a vacuum chamber 44 between one of the substrates 15A or 15B and the enclosure. Unlike the enclosure 30 or the enclosure 60, the enclosure 45 has a well on the opposite surface facing the one substrate. It is not necessary to have a concavo-convex shape for forming.
  • the cell culture container in the third embodiment has a desired uneven shape on one substrate only by applying a negative pressure from the outside to one substrate. It is comprised so that it may be formed.
  • the cell subculture method in the third embodiment is different from the first embodiment only in the principle of forming an uneven shape on one substrate of the cell culture container in the step (B). It is the same.
  • the enclosure 45 provided in the cell culture container in the third embodiment and the subculture system in the third embodiment is provided. Is used to form an uneven shape on one substrate of the cell culture vessel.
  • FIG. 5C shows an upper plan view of one substrate 15B of the cell culture container 40 in a modification of the third embodiment
  • FIG. 5D shows a side cross-sectional view taken along the line BB.
  • one substrate 15B has a sharp bottom end of a recess formed by applying a negative pressure to one substrate 15B.
  • the stretchable portion 41b can be gradually changed in thickness, and specifically, can be processed so that the portion forming the bottom end portion of the recess becomes the thinnest.
  • FIG. 6A shows an upper plan view of one substrate 15C of the cell culture vessel 50 used in a further modification of the third embodiment
  • FIG. 6B shows a side sectional view in the AA section.
  • FIG. 6E is a side cross-sectional view of a cell culture vessel 50 used in a further modification of the third embodiment in a cross section corresponding to the AA cross section of FIG.
  • One substrate 15C includes a hard portion 52 that forms a plurality of openings, and a stretchable portion 51 that extends into each opening of the hard portion.
  • the stretchable part 51 in the frame extends to form a concave 53 (microwell) uneven shape on one substrate 15C (for example, FIG. 6D).
  • the hard part 52 and the expansion / contraction part 51 can be designed according to the desired shape and arrangement of the dent 53, and when the hard part 52 and the expansion / contraction part 51 are arranged in a grid pattern as shown in FIGS. 5A and 5B, the dent is It is formed in a grid shape.
  • the stretchable part 51 is made of a stretchable material, and the stretchable part 51 can be pulled out of the cell culture container 50 by a constant negative pressure.
  • the hard part 52 may be the same as or different from the stretchable part 51 regardless of the material, as long as the hard part 52 has a hardness that does not substantially cause a shape change due to a certain negative pressure.
  • the hard portion 52 is made of the same stretchable material as that of the stretchable portion 51, but has a thickness that does not substantially cause a shape change due to a certain negative pressure. In this way, when a negative pressure is applied to the cell culture container 50 from the outside of the one substrate 15, the one substrate 15 ⁇ / b> C can form a recess 53 in each stretchable part 51.
  • the cell culture container 50 used in the further modification of the third embodiment forms an uneven shape on one substrate 15C and releases the negative pressure only by applying a negative pressure from the outside to one substrate 15C. This is advantageous over the cell culture vessel in the first embodiment that requires a complex-shaped enclosure in that the uneven shape can be eliminated.
  • FIG. 6C is a side cross-sectional view corresponding to FIG. 6B of one substrate 15D of the cell culture container in a further modification of the third embodiment.
  • the stretchable part 51 gradually changes the hardness of the film, for example, from the viewpoint of sharpening the bottom end of the formed microwell. Specifically, the hardness of the film gradually decreases from 51b to 51a, and the portion forming the bottom end of the microwell (for example, 51a) can be processed to have the lowest hardness. In this way, when a negative pressure is applied to the cell culture container from the outside of one substrate 15 ⁇ / b> D, the one substrate 15 ⁇ / b> D can form a recess 53 in the stretchable part 51.
  • substrate 15D which has the hard part 52 and the expansion-contraction part 51 can be easily produced by well-known methods, such as an insert molding method, if it is those skilled in the art.
  • the concave / convex shape is eliminated by forming a concave / convex shape on one substrate 15D and releasing the negative pressure only by applying a negative pressure from the outside to one substrate 15D. This is advantageous over the cell culture vessel in the first embodiment that requires a complex-shaped enclosure.
  • the cell culture container in the fourth embodiment is the same as the cell culture container 10 in the first embodiment.
  • a cell culture system 100 according to the fourth embodiment is a culture system used for culturing adherent cells. Although it does not specifically limit as an adherent cell, Adherent cells, such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell, are mentioned,
  • the cell culture system 100 which is 4th embodiment is a pluripotent stem cell, For example, it can be used for culturing embryonic stem cells (ES cells) and inducible pluripotent stem cells (iPS cells).
  • ES cells embryonic stem cells
  • iPS cells inducible pluripotent stem cells
  • a cell culture system 100 includes a cell culture container 10 and a pressing body 18.
  • a pressing body 18 is provided outside one substrate 15 of the cell culture container 10, and the cell culture system 10 is configured by the cell culture container 10 and the pressing body 18. .
  • adherent cells are seeded and adhered to at least one of the one substrate 15 and the other substrate 20, for example, the cell adhesion surface 20a formed by the other substrate 20. It can be cultured. The cells may be cultured while being adhered to the cell adhesion surface 15a of one substrate 15 and the cell adhesion surface 20a of the other substrate 20, and in this case, the cultured cells are cultured on one substrate 15 and the other substrate. Although it adheres to 20, the cells adhered to any cell adhesion surface can be peeled off using the pressing body 18.
  • the number of the pressing bodies 18 provided outside the one substrate 15 may be one or plural.
  • the plurality of culture chambers 17 may be pressed at a time by a single pressing body, or a plurality of presses installed corresponding to each culture chamber 17.
  • Each culture chamber 17 may be individually pressed by the body 18.
  • the pressing body 18 is installed outside the one substrate 15 of the cell culture container 10, and is pressed by pressing body operating means 18 A that operates the pressing body 18 toward the culture chamber 17.
  • the substrate 15 can be pressed toward the culture chamber 17. Thereby, one substrate 15 can be pushed into the culture chamber 17.
  • the pressing body 18 may be operated so as to push the one substrate 15 into the culture chamber 17 to the extent that it contacts the other substrate 20.
  • the pressing body 18 has a pressing surface smaller than the planar shape of each culture chamber 17 and presses a part of one substrate 15 corresponding to the planar shape of the entire culture chamber 17. be able to. Thereby, only the cells adhered to the pressed portion of one substrate 15 (or the portion corresponding to the pressed portion in the other substrate 20) can be peeled in a region-specific manner.
  • the shape of the pressing surface of the pressing body 18 is not particularly limited, but may be a circle or a rectangle.
  • the pressing surface of the pressing member 18 in a cell culture system for culturing pluripotent stem cells, a circular pressing surface diameter of about 0.4mm ⁇ 3.4mm (0.12mm 2 ⁇ 9mm 2 approximately in the area) It can be.
  • a cell can be exfoliated area-selectively, for example, each colony which a pluripotent stem cell forms during adhesion culture can be selectively exfoliated.
  • the pressing surface of the pressing body 18 is rectangular, it is easier to increase the shear stress than the circular shape.
  • the pressing surface of the pressing body 18 may be a planar shape or a curved surface shape, for example, a spherical shape.
  • the whole shape of the press body 18 is not specifically limited, For example, it can be set as a block shape. Therefore, in one preferable embodiment, the pressing body 18 is a block. From the viewpoint of reducing the risk of one substrate or the other substrate being broken, it is preferable that the pressing surface of the pressing body 18 has rounded corners.
  • the pressing body operating means 18A can preferably scrape and peel off the cells adhered to the cell adhesion surface by operating the pressing body 18 with the one substrate 15. In this case, the pressing body operating means 18A operates the pressing body 18 so that the cells are sandwiched between the one substrate 15 and the other substrate 20 and scraped off.
  • the pressing body operating means 18A is not particularly limited.
  • the pressing body 18 can be operated along the cell adhesion surface 20a of the other substrate 20 in a state where the one substrate 15 and the other substrate 20 are in contact with each other. Yes (FIGS. 19A-19D).
  • the operation of the pressing body 18 may be a simple straight movement, or may be accompanied by a movement such as a rotational movement or a reciprocating movement.
  • the cell culture system of the present embodiment includes a pressing body operating means that operates the pressing body and scrapes off the cells adhered to the cell adhesion surface with the one substrate 15. Further, when the cells adhered to the cell adhesion surface are scraped off by the one substrate 15, the pressing body 18 can be fixed and the culture vessel 10 can be operated.
  • the cell culture system of the fourth embodiment is provided with a sealed container, a sealed container, a door for putting the cell culture container into and out of the sealed container, a heating unit for heating the sealed container, and a temperature within the sealed container.
  • a temperature control unit that controls the temperature in the sealed container by controlling the heating unit based on the above may be further provided.
  • the cell culture system of the fourth embodiment may also include a cell culture container mounting table.
  • the cell culture system may further include a humidifying unit that humidifies the inside of the sealed container, and a humidity control unit that controls the humidity in the sealed container by controlling the humidifying unit based on the humidity in the sealed container.
  • the cell culture system according to the fourth embodiment includes at least one cell culture container 10.
  • the cell culture container 10 may be disposable, and the cell culture container 10 can be discarded and replaced with a new cell culture container 10 after use.
  • the cell culture vessel provided in the cell culture system of the fourth embodiment is preferably a closed cell culture vessel. Therefore, the cell subculture system of the present invention is preferably a closed cell culture system.
  • FIGS. 19A to 19D the operation of the cell culture system of the fourth embodiment is illustrated using the AA cross section of FIG. 16 as a sample.
  • adherent cells are seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20a formed by the other substrate 20. it can.
  • one substrate is pressed toward the culture chamber by the pressing body and pulled into the cell culture container, and the pressing body operating means is operated to move the cells by the one substrate. Cells can be detached by scraping.
  • FIG. 19A cells are cultured on the cell adhesion surface 20a of the other substrate 20.
  • the culture chamber 17 is filled with the medium 1.
  • FIG. 19B one substrate 15 is pressed by the pressing body 18 and pushed toward the culture chamber 17.
  • the volume of the culture chamber 17 varies, but the increase in the internal pressure in the culture chamber 17 can be suppressed by the other substrate not pressed by the pressing body bulging outward.
  • the pressing of the substrate by the pressing body may be performed while extracting an excess medium.
  • the retracted pressing body 18 is operated by the pressing body operating means 18A, and the cells are scraped off through one of the substrates.
  • the cell culture medium may be fed to remove the cells together with the detachment.
  • FIG. 19D only the scraped cells are peeled off from the substrate and collected or removed.
  • the cell culture system according to the fourth embodiment can be used when a specific cell, for example, a defective pluripotent stem cell colony is detached and removed from the cell adhesion surface.
  • a pressing body 18 ′ is provided outside the other substrate 20.
  • the pressing body 18 ′ is different from the pressing body 18 only in that it is installed outside the other substrate 20, and the rest is the same as the pressing body 18 of the cell culture system of the fourth embodiment.
  • the pressing body 18 and the pressing body 18 ′ may have different shapes, for example, the shape of the pressing surface.
  • the pressing surface of the pressing body 18 ′ has a shape corresponding to the planar shape of the culture chamber 17. May be.
  • the other substrate 20 of the cell culture container is made of a stretchable material like the one substrate 15, the pressing body 18 ′ faces the other substrate 20 toward the culture chamber 17. Can be pushed in. That is, in the modification of the cell culture system of the fourth embodiment, the other substrate 20 of the cell culture vessel 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17.
  • the cell culture system according to the fourth embodiment It is.
  • the risk of the substrate 15 and the other substrate 20 being broken can be reduced.
  • the substrate on the side that is not pressed by the pressing body bulges outward. An increase in internal pressure in the culture chamber can be prevented.
  • the pressing body 18 can be driven by the pressing body operating means 18A to scrape the cells from the cell adhesion surface.
  • the pressing body 18 ′ is driven by the pressing body operating means 18A ′, and the pressing body operating means 18A ′ preferably contacts one substrate 15 and the other substrate 20.
  • the other substrate 20 can be pushed toward the culture chamber 17 by the pressing body 18 ′ to such an extent that it can be moved.
  • the pressing body operating means 18A ′ can be driven parallel to the other substrate 20 when the cells adhered to the cell adhesion surface are sandwiched between the one substrate 15 and the other substrate 20 and peeled off.
  • the other substrate 20 may be stationary while being pushed toward the culture chamber 17.
  • the pressing body 18 ′ is driven by the pressing body operating means 18 A ′ and pushed into the culture chamber 17, thereby The distance which pushes the press body 18 installed in the outward direction can be reduced.
  • FIGS. 20A to 20D a cell culture system 100 according to a fifth embodiment will be described with reference to FIGS. 20A to 20D.
  • the cell culture system of the fifth embodiment shown in FIGS. 20A to 20D is only different in the shape of the pressing body 18 and the driving method, and the other configuration is the cell of the fourth embodiment shown in FIGS. It is the same as the culture system. 20A to 20D, the same parts as those of the cell culture system of the fourth embodiment shown in FIGS.
  • the pressing body 18 is disposed on the outer side on the one substrate 15 side in the cell culture system of the present invention.
  • the pressing body 18 has a pressing surface having a shape corresponding to the planar shape of each culture chamber 17 (for example, FIG. 16), and one substrate 15 corresponding to the planar shape of the entire culture chamber 17 faces the culture chamber 17. Can be pressed.
  • the pressing body 18 presses one of the substrates 15 so that the one substrate 15 and the other substrate 20 are 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.00 mm. It is possible to approach the distance of 5 mm, more preferably, the approaching distance can be freely adjusted, and further preferably, the culture is performed by a distance necessary for bringing one substrate 15 into contact with the other substrate 20. It can be moved toward the chamber 17. By doing in this way, strong shearing force can be generated by feeding liquid to the cells adhered to one substrate 15 or the other substrate 20, and the cells can be detached from the cell adhesion surface.
  • FIGS. 20A to 20D the operation of the cell culture system of the present invention is illustrated using the AA cross section of FIG. 16 as a sample.
  • adherent cells can be seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20 a formed by the other substrate 20.
  • one substrate is pressed toward the culture chamber by the pressing body and drawn into the cell culture container, and the cell is collected by feeding the cell recovery solution to the culture chamber. Can be peeled off.
  • FIG. 20A cells are cultured on the cell adhesion surface 20a of the other substrate 20.
  • the culture chamber 17 is filled with the medium 1.
  • one substrate 15 is pressed by the pressing body 18 and pushed toward the culture chamber 17.
  • the one substrate 15 and the other substrate 20 are brought close to a distance of, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm.
  • the pressing of the substrate by the pressing body can be performed while removing the excess medium.
  • the liquid supply means for supplying the cell recovery liquid is operated, and the cell recovery liquid is operated. Is fed to the flow path 13 and the culture chamber 17. Then, the shear stress generated on the cell culture surface 20a due to the liquid feeding increases, and as shown in FIG. 20D, the cells are peeled off from the cell adhesion surface 20a corresponding to the pressing surface by the pressing body 18 to collect the cells. it can. When the pressing by the pressing body 18 is finished, it can be performed while feeding the culture medium in order to prevent a decrease in internal pressure. By doing in this way, in the cell culture system 100 which is 5th embodiment, a cell can be peeled from the whole cell culture surface.
  • the cell recovery solution can be, for example, a cell culture medium or a physiological saline such as phosphate buffered saline.
  • the shear stress generated when the viscous liquid flows in the cylinder can be expressed by the following equation. ⁇ Wherein, a is a constant, ⁇ indicates shear stress (mPa), ⁇ indicates the viscosity coefficient (mPa ⁇ min) of the liquid to be fed, Q indicates the flow rate ( ⁇ L / min), ⁇ Indicates the circumference, and r indicates the radius (mm) of the cylinder. ⁇
  • FIG. 21 is a diagram showing the flow rate of the viscous fluid flowing in the cylinder having various radii and the shear stress generated by the viscous fluid on the side wall.
  • the viscosity coefficient ⁇ of the viscous liquid is 0.685 mPa ⁇ sec
  • the relationship between the flow rate Q and the shear stress ⁇ when the simulation is performed when the cylindrical radii are 1 mm, 2 mm, and 3 mm is shown.
  • the radius of the cylinder decreases, the shear stress on the cylinder side wall by the viscous fluid increases rapidly.
  • the shear stress generated on the side wall of the flow path increases.
  • the shear stress generated on the cell culture surface of the culture chamber 17 can be increased.
  • the cell adhesion surface corresponding to the portion drawn by the pressing body 18.
  • the shear stress generated by the fluid increases at 20a, and thus the cells can be detached from the cell adhesion surface 20a (FIGS. 20A to 20D).
  • the liquid feeding is performed from the inlet 11, and the pressing body 18 is directed toward the culture chamber 17 so as to increase the shearing force generated by the liquid feeding on the cell adhesion surface 20 a corresponding to the portion drawn by the pressing body 18. Can be pushed in.
  • one of the substrates is pressed toward the culture chamber by the pressing body and pulled into the cell culture container, and the cell recovery solution is fed to the culture chamber.
  • a method for detaching cultured cells comprising the step of detaching cells.
  • the cell culture system according to the fifth embodiment can be used, for example, at the time of cell passage, for example, when the whole cell adhered to the cell culture surface is peeled and recovered.
  • a pressing body 18 ′ is provided outside the other substrate 20.
  • the pressing body 18 ′ is different from the pressing body 18 only in that it is installed outside the other substrate 20, and the rest is the same as the pressing body 18 of the cell culture system of the fourth embodiment.
  • the pressing body 18 and the pressing body 18 ′ may have different shapes, for example, the shape of the pressing surface.
  • the pressing surface of the pressing body 18 ′ has a shape corresponding to the planar shape of the culture chamber 17. May be.
  • the other substrate 20 of the cell culture container 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17.
  • the pressing body 18 ′ can push the other substrate 20 toward the culture chamber 17 when peeling the cells adhered to the cell adhesion surface. Accordingly, either or both of the pressing body 18 and the pressing body 18 ′ can be pushed toward the culture chamber 17. At this time, the one substrate 15 and the other substrate 20 are brought close to a distance of, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm. In order to prevent an increase in the internal pressure in the culture chamber, the pressing of the substrate by the pressing body can be performed while removing the excess medium. Subsequent operations are the same as those of the cell culture system according to the fifth embodiment.
  • the liquid feeding means for feeding the cell recovery solution is operated, and the cell recovery solution is supplied to the channel 13 and the culture chamber 17. Deliver liquid. Then, the shear stress generated on the cell culture surface 20a due to the liquid feeding increases, and the cells can be separated from the cell adhesion surface 20a corresponding to the pressing surface by the pressing body 18 to collect the cells.
  • the pressing by the pressing body 18 is finished, it can be performed while feeding the culture medium in order to prevent a decrease in internal pressure. By doing in this way, in the modification of the cell culture system which is 5th embodiment, a cell can be peeled from the whole cell culture surface.
  • the other substrate 20 may be pressed into the culture chamber 17 by the pressing body 18 ′. Pressing by either or both of the pressing body 18 and the pressing body 18 ′ can be performed so as to increase the shearing force generated by liquid feeding on the cell adhesion surface 20 a.
  • the pressing body 18 when the pressing body 18 is provided outside the one substrate 15 and outside the other substrate 20, respectively, as shown in FIG. 24E, the one substrate 15 and the other substrate 20 are sandwiched between the culture chambers 17, respectively. Can be pushed toward the culture chamber 17. In this way, the deformation amount of the stretchable material of one substrate 15 and the other substrate 20 can be reduced (for example, halved) compared to the case where only one substrate 15 is a stretchable material. And the risk of the other substrate 20 being broken can be reduced.
  • FIGS. 23A to 23D a cell culture system 100 according to a sixth embodiment will be described with reference to FIGS. 23A to 23D.
  • the cell culture system of the sixth embodiment shown in FIGS. 23A to 23D is only different in the driving method of the pressing body 18, and the other configuration is the cell culture system of the fourth embodiment shown in FIGS. It is the same.
  • FIG. 23A to FIG. 23D the same parts as those of the cell culture system of the fourth embodiment shown in FIGS.
  • the pressing body 18 has the same shape as that of the cell culture system 100 of the fourth embodiment, and the one substrate 15 corresponding to the planar shape of the entire culture chamber 17 is provided. A part can be pressed.
  • FIG. 23A to FIG. 23D illustrate the operation of the cell culture system of the present invention using the AA cross section of FIG. 16 as a sample.
  • adherent cells are seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20a formed by the other substrate 20. it can.
  • FIG. 23A cells are cultured on the cell adhesion surface 20a of the other substrate 20.
  • the culture chamber 17 is filled with the medium 1.
  • FIG. 23B a part of one substrate 15 is pressed by the pressing body 18 and pushed toward the culture chamber 17.
  • a part of the one substrate 15 and the other substrate 20 are, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm. Move closer.
  • the pressing of the one substrate 15 by the pressing body 18 can be performed while extracting an excess medium.
  • the cell culture system of the sixth embodiment does not grind the cells. Therefore, in the cell culture system of the sixth embodiment, specific cells adhering to the cell culture surface may be detached, and then the detached cells may be collected and passaged. Of course, specific cells detached by the cell culture system of the sixth embodiment, for example, defective pluripotent stem cell colonies, may be removed.
  • the modified example of the cell culture system of the sixth embodiment is different only in the driving method of the pressing body 18, and the other parts are the same as the modified example of the cell culture system of the fourth embodiment. That is, in the modification of the cell culture system of the sixth embodiment, the other substrate 20 of the cell culture container 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the cell culture system according to the sixth embodiment, in which the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17. It is. Therefore, in the modification of the cell culture system of the sixth embodiment, only the driving method of the pressing body 18 will be described.
  • the pressing body 18 presses a part of one substrate 15 to enter the culture chamber 17. Push in. At this time, a part of the one substrate 15 and the other substrate 20 are, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm. Move closer. At this time, although not particularly limited, the pressing body 18 ′ may be pushed into the culture chamber 17 (FIG. 24C). By doing in this way, the risk that one board
  • the modification of the cell culture system of the sixth embodiment does not grind the cells. Therefore, in the modification of the cell culture system of the sixth embodiment, specific cells adhered to the cell culture surface may be detached, and then the detached cells may be collected and passaged. Of course, specific cells detached in the modification of the cell culture system of the sixth embodiment, for example, defective pluripotent stem cell colonies, may be removed.
  • FIGS. 20A to 20D The configuration of the cell culture system of the seventh embodiment is the same as the cell culture system of the fifth embodiment shown in FIGS. 20A to 20D. The only difference is that in FIG. 20A or FIG. 20B, preferably FIG. 20B, a cell detachment solution is sent when cells are further detached in order to weaken cell adhesion. That is, in the cell culture system of the fifth embodiment, one of the substrates is pressed toward the culture chamber by a pressing body and pulled into the cell culture container, and the cell is collected by feeding the cell recovery solution to the culture chamber. Although it can be peeled off, the cell culture system of the seventh embodiment further includes weakening cell adhesion by feeding a cell peeling solution before feeding the cell recovery solution.
  • cell detachment solutions are known as cell detachment solutions, and those skilled in the art can select an appropriate cell detachment solution as the cell detachment solution.
  • the cell detachment solution may be a cell culture medium or physiological saline containing at least one of a proteolytic enzyme such as trypsin and a divalent metal ion chelator.
  • a proteolytic enzyme such as trypsin and a divalent metal ion chelator.
  • what does not contain serum can be used.
  • the amount of the cell detachment liquid to be used can be reduced. Particularly, in the case of mass culture of cells, a large amount of expensive chemical solution is used, so the merit of cost reduction by reducing the amount of chemical solution is great.
  • the cell culture system of the seventh embodiment one substrate is pressed toward the culture chamber by the pressing body and pulled into the cell culture container, and then the cell detachment liquid is fed.
  • a method of exfoliating cultured cells comprising weakening cell adhesion and then exfoliating the cells by delivering a cell recovery solution to the culture chamber.
  • the cell culture system according to the seventh embodiment can be used, for example, at the time of cell passage, for example, when the whole cell adhered to the cell culture surface is peeled and collected.
  • the modification of the cell culture system of the seventh embodiment is different only in that the pressing body 18 ′ and the pressing body drive mechanism 18A ′ are further provided (FIG. 24D), and other configurations are the cells of the seventh embodiment.
  • the other substrate 20 of the cell culture container 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the cell culture system according to the seventh embodiment, in which the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17. It is.
  • the cells described in the seventh embodiment when the cells are peeled off from the cell adhesion surface in the cell culture vessel, the cells described in the seventh embodiment while pushing the pressing body 18 and the pressing body 18 ′ into the culture chamber 17, as shown in FIG. 24E. You may perform the process of liquid feeding of peeling liquid. By doing in this way, the risk that one board
  • the modified example of the cell culture system of the seventh embodiment can be used, for example, when the whole cell adhered to the cell culture surface is peeled and collected, for example, when the cell is passaged.
  • FIGS. 20A to 20D a cell culture system 100 according to an eighth embodiment will be described with reference to FIGS. 20A to 20D.
  • the cell culture system of the eighth embodiment is different only in the driving method of the pressing body 18, and other configurations are the same as those of the cell culture system of the sixth embodiment shown in FIGS. 23A to 23D.
  • the driving method of the pressing body in the cell culture system 100 of the eighth embodiment is different from the cell culture system of the fifth embodiment only in that the pressing body 18 performs high-frequency vibration. That is, in the cell culture system of the fifth embodiment, one of the substrates is pressed toward the culture chamber by a pressing body and pulled into the cell culture container, and the cell is collected by feeding the cell recovery solution to the culture chamber.
  • the cell culture system according to the eighth embodiment further includes weakening cell adhesion by applying high-frequency vibration to the cell adhesion surface of the other substrate before feeding the cell recovery solution.
  • the pressing body 18 may have the same shape as the cell culture system 100 of the fifth embodiment shown in FIGS. 20A to 20D.
  • FIGS. 20A to 20D the operation of the cell culture system of the present invention is illustrated using the AA cross section of FIG. 16 as a sample.
  • adherent cells can be seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20 a formed by the other substrate 20.
  • FIG. 20A cells are cultured on the cell adhesion surface 20a of the other substrate 20.
  • the culture chamber 17 is filled with the medium 1.
  • one substrate 15 is pressed by the pressing body 18 and pushed toward the culture chamber 17.
  • the one substrate 15 and the other substrate 20 are brought close to a distance of, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm.
  • the pressing of the substrate by the pressing body can be performed while removing the excess medium.
  • the pressing body driving unit 18A is driven in a state in which one substrate 15 is pressed by the pressing body 18 and pulled into the culture chamber 17.
  • high-frequency vibration is generated from the pressing body 18.
  • high frequency vibration is transmitted to the cell adhesion surface 20a, and the adhesion between the cell adhesion surface 20a and the cells is weakened.
  • the high-frequency vibration may be applied before the start of liquid feeding or may be applied simultaneously with the liquid feeding.
  • the cell recovery liquid is supplied to the channel 13 and the culture chamber 17 by a liquid supply means for supplying the cell recovery liquid. Then, as shown in FIG.
  • the cells can be separated from the cell adhesion surface 20 a corresponding to the pressing surface by the pressing body 18 by the shear stress generated on the cell culture surface 20 a by the liquid feeding, and the cells can be collected.
  • the pressing by the pressing body 18 is finished, it can be performed while feeding the culture medium in order to prevent a decrease in internal pressure.
  • a cell can be peeled from the whole cell culture surface.
  • one substrate is pressed toward the culture chamber by the pressing body and drawn into the cell culture container, and then before the cell recovery liquid is fed. And applying a high frequency vibration to the cell adhesion surface of the other substrate to weaken the cell adhesion, and then peeling the cells by sending a cell recovery solution to the culture chamber.
  • Ultrasonic waves can be used as the high-frequency vibration.
  • a piezoelectric element a plastic ultrasonic welder, a commercially available ultrasonic generator for cell disruption, or an ultrasonic generator for an ultrasonic cleaner is used. It can be generated by using as a high frequency vibration source.
  • the high frequency is, for example, preferably 20 Hz to 10 GHz, more preferably 16 kHz to 10 GHz, further preferably 16 kHz to 1 GHz, and further preferably 20 kHz to 100 kHz.
  • the high frequency vibration of 70 kHz can be concentrated on a certain circular portion (for example, ⁇ 2 mm) to give the high frequency vibration.
  • the cell culture system of the eighth embodiment can be used, for example, when peeling and collecting the entire cells adhered to the cell culture surface, for example, at the time of cell passage.
  • the cell culture system of the eighth embodiment presses a part of one substrate 15 corresponding to the planar shape of the entire culture chamber 17 toward the culture chamber 17 (for example, FIG. 23B), and presses in that state.
  • a high frequency vibration is generated from the body 18 to weaken the adhesion between the cell adhesion surface 20a adhering to the cell culture surface and the specific cell, and then the detached cell may be collected by feeding a cell collection solution.
  • Cells that have exfoliated in a region-specific manner may be collected and passaged, or exfoliated cells (for example, defective pluripotent stem cell colonies) may be removed.
  • the modification of the cell culture system of the eighth embodiment is different only in that the pressing body 18 ′ and the pressing body drive mechanism 18A ′ are further provided, and the other configurations are the same as those of the cell culture system of the eighth embodiment. It is. That is, in the modification of the cell culture system of the eighth embodiment, the other substrate 20 of the cell culture vessel 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the cell culture system according to the eighth embodiment, in which the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17. It is.
  • the cell recovery solution feeding step described in the eighth embodiment while pushing the pressing body 18 and the pressing body 18 ′ into the culture chamber 17 is performed. May be performed (FIG. 24E).
  • the modified example of the cell culture system of the eighth embodiment can be used, for example, when the whole cell adhered to the cell culture surface is peeled and recovered, for example, at the time of cell passage.
  • the modification of the cell culture system of 8th embodiment presses a part of one board
  • a cell culture system 100 according to a ninth embodiment will be described with reference to FIGS. 22A to 22F.
  • the cell culture system of the ninth embodiment is different only in that it further includes a high-frequency vibration generating unit 19 that applies high-frequency vibration to the cell adhesion surface 20a, and the other configurations are the fifth configuration shown in FIGS. 20A to 20D.
  • This is the same as the cell culture system of the embodiment. 22A to 22F, the same parts as those in the cell culture system of the fifth embodiment shown in FIGS. 20A to 20D are denoted by the same reference numerals, and description thereof is omitted.
  • the high-frequency vibration generator 19 is installed outside the other substrate 20 of the cell culture system of the present invention.
  • the high-frequency vibration generating unit 19 has a pressing surface having a shape corresponding to the planar shape (for example, FIG. 16) of each culture chamber 17, and can apply high-frequency vibration to the entire cell adhesion surface 20a.
  • the high frequency vibration generation part 19 has a pressing surface smaller than the planar shape of each culture chamber 17, and can weaken the cell adhesion of the cell adhesion surface 20a area-specifically by high frequency vibration.
  • the cell culture system of the ninth embodiment further includes weakening cell adhesion by applying high-frequency vibration to the cell adhesion surface of the other substrate before feeding the cell recovery solution.
  • the high-frequency vibration generator 19 can use ultrasonic waves as the high-frequency vibration, and the ultrasonic waves are, for example, piezoelectric elements, plastic ultrasonic welders, commercially available ultrasonic generators for cell disruption, or ultrasonic cleaners.
  • the ultrasonic generator can be generated as a high frequency vibration source.
  • the high frequency is, for example, preferably 20 Hz to 10 GHz, more preferably 16 kHz to 10 GHz, further preferably 16 kHz to 1 GHz, and further preferably 20 kHz to 100 kHz.
  • the high frequency vibration of 70 kHz can be concentrated on a certain circular portion (for example, ⁇ 2 mm) to give the high frequency vibration.
  • the high-frequency vibration can peel the cells when the high-frequency vibration generation source is brought close to or brought into contact with one substrate 15 or the other substrate 20 having the cell adhesion surface from the outside of the culture vessel. Cells that have been detached from the cell adhesion surface by applying high-frequency vibration can be cultured well thereafter. High frequency vibration can also be used to weaken the adhesion between cells and the cell adhesion surface.
  • the high-frequency vibration can be applied to one substrate 15 or the other substrate 20 before being pressed against one substrate 15 by the pressing body 18. From the viewpoint of peeling cells from the cell adhesion surface by high-frequency vibration, the cell adhesion surface is preferably formed of a stretchable material in order to reduce the risk of breakage due to friction and heat generated by ultrasonic vibration.
  • the high-frequency vibration generator 19 applies high-frequency vibration to the plurality of culture chambers 17 by one high-frequency vibration generator 19 when the cell culture vessel 10 includes a plurality of culture chambers 17. Alternatively, high frequency vibration may be applied to each culture chamber 17 individually.
  • FIG. 22B to FIG. 22E the operation of the cell culture system of the present invention is illustrated using the AA cross section of FIG. 16 as a sample.
  • adherent cells can be seeded and cultured on at least one of the one substrate 15 and the other substrate 20, for example, on the cell adhesion surface 20 a formed by the other substrate 20.
  • FIG. 22B cells are cultured on the cell adhesion surface 20a of the other substrate 20.
  • the culture chamber 17 is filled with the medium 1.
  • the high-frequency vibration generating unit 19 is installed outside the other substrate 20 and applies high-frequency vibration to the other substrate 20 to weaken the adhesion between the cell adhesion surface 20a and the cells. High frequency vibration is preferably applied for about 0.5 seconds.
  • FIG. 22C one substrate 15 is pressed by the pressing body 18 and pushed toward the culture chamber 17.
  • the one substrate 15 and the other substrate 20 are brought close to a distance of, for example, 0.1 mm to 2 mm, preferably 0.1 mm to 1 mm, more preferably 0.1 mm to 0.5 mm.
  • the pressing of the substrate by the pressing body can be performed while removing the excess medium.
  • FIG. 22D in a state where one substrate 15 is pressed by the pressing body 18 and pulled into the culture chamber 17, the liquid supply means for supplying the cell recovery liquid is operated, and the cell recovery liquid is operated. Is fed to the flow path 13 and the culture chamber 17. Then, the shear stress generated on the cell culture surface 20a by liquid feeding increases, and as shown in FIG.
  • the cells are detached from the cell adhesion surface 20a corresponding to the pressing surface by the pressing body 18 and the cells can be collected. it can.
  • the pressing by the pressing body 18 is finished, it can be performed while feeding the culture medium in order to prevent a decrease in internal pressure.
  • a cell can be peeled from the whole cell culture surface.
  • the high-frequency vibration generator 19 may be lowered from the other substrate 20 after the oscillation process and slightly separated, or may be oscillated only during the oscillation process while being kept in contact.
  • one substrate is pressed toward the culture chamber by the pressing body and pulled into the cell culture container, and then before the cell recovery liquid is fed. And applying a high frequency vibration to the cell adhesion surface of the other substrate to weaken the cell adhesion, and then peeling the cells by sending a cell recovery solution to the culture chamber.
  • the cell culture system of the ninth embodiment can be used, for example, at the time of cell passage, for example, when the entire cell adhered to the cell culture surface is peeled and recovered.
  • one substrate 15 corresponding to the planar shape of the entire culture chamber 17 is pressed toward the culture chamber 17 to weaken cell adhesion in a region-specific manner by high-frequency vibration. Specific cells adhering to the cell culture surface may be detached, and then the detached cells may be collected and passaged, or the detached cells, for example, defective pluripotent stem cell colonies may be removed Good.
  • the modification of the cell culture system of the ninth embodiment is different only in that the high-frequency vibration generating unit 19 has a function as the pressing body 18 ', and other configurations are the cells of the ninth embodiment.
  • the other substrate 20 of the cell culture container 10 is made of a stretchable material, and further includes a pressing body 18 ′ outside the other substrate 20.
  • the pressing body 18 ′ installed outside the substrate 20 is a pressing body that presses the other substrate 20 toward the culture chamber 17 and retracts it into the culture chamber 17.
  • the cell culture system according to the ninth embodiment It is.
  • the cell recovery solution is passed through the flow path 13 and the culture chamber while pushing both the pressing body 18 and the high-frequency vibration generator 19 into the culture chamber 17.
  • the cells adhered to the cell adhesion surface in the culture chamber can be peeled off.
  • the high-frequency vibration is generated in the high-frequency vibration generator 19 while pushing both the pressing body 18 and the high-frequency vibration generator 19 into the culture chamber 17. May be generated.
  • the modification of the cell culture system according to the ninth embodiment can be used, for example, when the whole cell adhered to the cell culture surface is peeled and collected, for example, when the cells are passaged. Further, in the modified example of the cell culture system of the ninth embodiment, one substrate 15 corresponding to the planar shape of the entire culture chamber 17 is pressed toward the culture chamber 17 and cell adhesion is performed region-specifically by high-frequency vibration. The specific cells adhering to the cell culture surface can be detached by weakening, and then the detached cells can be collected and passaged, or the detached cells, for example, defective pluripotent stem cell colonies can be removed May be.
  • the cell culture container 70 of the tenth embodiment includes one substrate 71, the other substrate 72 arranged opposite to the one substrate 71, A side wall 73 that forms a culture chamber 75, an inlet 76 that allows a liquid to flow into the culture chamber 75, an outlet 77 that allows a liquid to flow out of the culture chamber 75, and a culture medium, which are interposed between the substrate 71 and the other substrate 72.
  • the chamber 75 includes a sub-side wall 74 that stands up from the other substrate 72 and has a height lower than that of the side wall 73.
  • the one substrate 71 and the other substrate 72 are the same as the one substrate 15 and the other substrate 20 of the cell culture container 10 of the first embodiment, respectively, the description regarding the one substrate 15 and the other substrate 20 is performed. Can also be applied to one substrate 71 and the other substrate 72.
  • One substrate 71 is formed of a stretchable material.
  • One substrate 71 is made of a stretchable material at least in a region corresponding to the planar shape of the culture chamber 75. Thus, one substrate 71 can be pressed toward the culture chamber 75 and pushed into the culture chamber 75. A portion of one substrate 71 pushed into the culture chamber 75 can abut on the sub-side wall 74 standing from the other substrate 72 in the culture chamber 75. When one substrate 71 is not pressed toward the culture chamber 75, the one substrate 71 is separated from the sub-side wall 74 and forms an integral space in the culture chamber 75.
  • the stretchable material is preferably transparent from the viewpoint of facilitating microscopic observation, and more preferably colorless and transparent. As such a stretchable material, for example, polydimethylsiloxane (PDMS) or silicone can be used.
  • One substrate 71 is preferably formed of silicone rubber. Thereby, flexibility and strength can be imparted to one substrate 71.
  • One substrate 71 is not particularly limited, and has a thickness of 0.01 mm to 0.3 mm, 0.05 mm to 0.1 mm, or 0.02 mm to 0.2 mm, for example.
  • the hardness of one substrate 71 can be 20 to 65, preferably 20 to 30 in terms of type A durometer hardness.
  • the thickness of one substrate 71 is preferably 0.05 mm to 0.3 mm.
  • a protective film for enhancing the strength of the one substrate 71 may be provided on the surface of the one substrate 71, particularly on the surface of the portion formed of the stretchable material.
  • the deformation of one substrate 71 causes a liquid flow in the culture chamber 75, and thus prevention of the deformation of the one substrate 71 is important from the viewpoint of managing the liquid flow in the culture chamber 75.
  • the material which forms a protective film is not specifically limited, For example, a polyethylene terephthalate (PET), a polystyrene, etc. are mentioned.
  • the thickness can be 0.2 mm, for example, and when using a polystyrene film as the protective film, the thickness can be 0.13 mm, for example.
  • the protective film is preferably transparent so as not to hinder observation through one substrate 71.
  • the thickness and hardness of the other substrate 72 may be the same as or different from those of the one substrate 71.
  • the other substrate 72 is preferably transparent from the viewpoint of facilitating microscopic observation.
  • the other substrate 72 is not particularly limited, but is preferably a gas permeable membrane from the viewpoint of maintaining the oxygen concentration and carbon dioxide concentration in the culture chamber 75.
  • a gas permeable membrane made of polystyrene is used as a gas permeable membrane for cell culture. Membranes are commercially available.
  • the gas permeable membrane plays an important role in supplying oxygen or the like to the culture medium in the culture chamber 75.
  • the other substrate 72 may be formed of a non-stretchable material, but may be formed of a stretchable material like the one substrate 71.
  • the volume fluctuation of the culture chamber 75 that occurs when the pressing body is pushed toward the one substrate 71, the other substrate 72 side is outward. It can be absorbed by swelling. Therefore, the risk of liquid leakage from the inlet 76 and the outlet 77 that can be sealed with a rubber stopper or the like can be suppressed.
  • the other substrate 72 is formed of a stretchable material
  • the other substrate 72 is preferably formed of silicone rubber. Thereby, flexibility and strength can be imparted to the other substrate 72.
  • a protective film that enhances the strength of the other substrate 72 may be provided on the surface of the other substrate 72. Since the description regarding the protective film is the same as that of the one substrate 71, the description thereof is omitted.
  • the other substrate 72 may be a member different from the side wall 73 and / or the sub-side wall 74, or may be a member integrally formed with the side wall 73 and / or the sub-side wall 74.
  • substrate 72 is a member different from the side wall 73 and / or the sub-side wall 74
  • substrate 72 is the side wall 73 and / or the sub-side wall 74 by a well-known joining mode (for example, adhesion
  • One substrate 71 may form a cell adhesion surface 710.
  • the inner surface of the culture chamber 75 other than the cell adhesion surface is preferably non-cell-adhesive, and may be coated with a cell non-adhesion for the purpose of preventing cell adhesion. Since the description regarding the cell non-adhesive coating is the same as that of the cell culture container 10 of the first embodiment, the description thereof is omitted.
  • the cell adhesion surface 720 of the other substrate 72 is preferably cell adhesion, and may be coated with a cell adhesion coating for the purpose of imparting cell adhesion.
  • the side wall 73 is not particularly limited, it can be made of plastic, for example, and can be made of polystyrene, polypropylene, polycarbonate, or acrylic, for example.
  • the side wall 73 can also be formed of silicone rubber. Thereby, flexibility and strength can be imparted to the side wall 73.
  • the side wall 73 is a peripheral wall surrounding the periphery of the culture chamber 75, and the culture chamber 75 is formed by a space surrounded by the one substrate 71, the other substrate 72, and the side wall 73.
  • the side wall 73 forms a cell culture container body together with the other substrate 72 and the sub-side wall 74.
  • the side wall 73 may be a member different from the other substrate 72 and / or the sub-side wall 74, or may be a member formed integrally with the other substrate 72 and / or the sub-side wall 74.
  • the side wall 73 is a member different from the other substrate 72 and / or the sub-side wall 74, the side wall 73 is connected to the other substrate 72 and / or the sub-side wall 74 by a known bonding mode (for example, adhesion by an adhesive). Can be joined.
  • the sub-side wall 74 stands up from the other substrate 72 in the culture chamber 75 and has a height H1 lower than the height H2 of the side wall 73.
  • a groove 78 extending from the inlet 76 to the outlet 77 is formed in the culture chamber 75 by the sub-side wall 74.
  • the height H ⁇ b> 1 of the sub-side wall 74 is lower than the height H ⁇ b> 2 of the side wall 73, there is a gap between one substrate 71 and the end of the sub-side wall 74 on the one substrate 71 side. That is, when one substrate 71 is not pressed, one substrate 71 is separated from the sub-side wall 74. Thus, a culture chamber 75 surrounded by one substrate 71, the other substrate 72, and the side wall 73 is formed in the cell culture container 70 as an integral space.
  • the height H1 of the sub-side wall 74 is not particularly limited as long as it is lower than the height H2 of the side wall 73.
  • the height H1 of the sub-side wall 74 is a distance from the surface of the other substrate 72 in contact with the culture chamber 75 to the end of the sub-side wall 74 on the one substrate 71 side, and the height H2 of the side wall 73 is This is the distance from the surface of the other substrate 72 in contact with the culture chamber 75 to the end of the side wall 73 on which the one substrate 71 is provided.
  • the height H1 of the sub-side wall 74 is preferably 0.5 to 2.0 mm.
  • the number of lay nozzles in the flow path 79 can be reduced to 2000 or less.
  • the flow of liquid in the flow path 79 (a wide flow from low speed to high speed) can be controlled in a laminar flow state. Controlling the flow of the liquid in the flow path 79 to a laminar flow state is important from the viewpoint of preventing the liquid from staying in the flow path 79.
  • the difference between the height H2 of the side wall 73 and the height H1 of the auxiliary side wall 74 can be adjusted as appropriate according to the volume of the culture chamber 75 necessary for cell culture.
  • the corner of the sub-side wall 74 against which the one substrate 71 is pressed is preferably chamfered.
  • the number of sub-side walls 74 is not particularly limited, and may be one or plural. In the present embodiment, five sub-side walls are provided. When a plurality of sub-side walls 74 are provided, their heights may be the same or different, but are preferably the same or substantially the same. In the present embodiment, the heights of the five sub-side walls are the same or substantially the same.
  • the side wall 73 has two side wall portions extending in the short direction of the cell culture container 70 and two side wall portions extending in the longitudinal direction of the cell culture container 70.
  • the sub-side wall 74 extended in the longitudinal direction of the cell culture container 70 so that it may not reach the other from one of the two side wall parts extended in the transversal direction of the cell culture container 70, and the cell culture container 70
  • the sub-side walls 74 extending in the longitudinal direction of the cell culture container 70 are alternately arranged so as not to reach one from the other of the two side wall portions extending in the short direction.
  • the space in the groove 78 is partially discontinuous by being blocked by the sub-side wall 74, but from one of the two side walls extending in the short direction of the cell culture vessel 70 to the other. So as not to reach between the sub-side wall 74 extending in the longitudinal direction of the cell culture vessel 70 and the other of the two side wall portions extending in the short direction of the cell culture vessel 70, and the cell culture vessel 70
  • the sub-side wall 74 extending in the longitudinal direction of the cell culture vessel 70 so as not to reach one from the other of the two side wall portions extending in the short direction of the cell, and extending in the short direction of the cell culture vessel 70 It is continuous between one of the two side walls.
  • a groove portion 78 extending from the inlet 76 to the outlet 77 and communicating with the inlet 76 and the outlet 77 is formed while reversing the extending direction.
  • the culture chamber 75 has a cell adhesion surface 710 or a cell adhesion surface 720 formed by at least one of the one substrate 71 and the other substrate 72, as shown in FIGS. 27, 28A, and 28B.
  • the number of the culture chambers 75 in this embodiment is one, the number of the culture chambers 75 in the cell culture container 70 is not specifically limited, A plurality may be sufficient.
  • each culture chamber 75 is connected in series by a flow path, like the cell culture container 10 of the first embodiment shown in FIG. May be.
  • each culture chamber 75 may include an independent inlet 76 and outlet 77.
  • the space in the culture chamber 75 can be divided into a first space in the groove 78 and a second space located closer to the one substrate 71 than the first space.
  • the groove part 78 has an opening part that opens toward the one substrate 71, and the first space and the second space are continuous through the opening part of the groove part 78.
  • the first space is blocked by the sub-side wall 74 and is partially discontinuous.
  • the second space is not blocked by the sub-side wall 74 and is entirely continuous.
  • an inlet 76 through which liquid flows into the culture chamber 75 and an outlet 77 through which liquid flows out from the culture chamber 75 are formed.
  • various solutions for example, cell culture solution, cell recovery solution, cell detachment solution, etc.
  • the inlet 76 is used as an inlet of the solution to be sent, and the outlet 77 is sent. Used as a solution outlet.
  • the inlet 76 and the outlet 77 are formed in the side wall 73, the inlet 76 is passed through the flow path 760 formed in the side wall 73, and the outlet 77 is passed through the flow path 770 formed in the side wall 73. It communicates with the outside of 70.
  • the place where the inlet 76 and the outlet 77 are formed is not particularly limited as long as the liquid can flow into the culture chamber 75 and the liquid can flow out of the culture chamber 75.
  • the inlet 76 and the outlet 77 are formed on the other substrate 72. May be.
  • the culture chamber 75 is an integral space. However, the one substrate 71 is pressed toward the culture chamber 75, and the sub-side wall is pressed.
  • the opening of the groove 78 is sealed by one substrate 71, and a flow path 79 communicating with the inlet 76 and the outlet 77 is formed (see FIG. 29C).
  • One end of the channel 79 communicates with the inlet 76, the other end of the channel 79 communicates with the outlet 77, and the inlet 76 and the outlet 77 communicate with each other through the space in the channel 79.
  • the space in the flow path 79 is cut off from the space around the flow path 79, and the flow path 79 is tubular.
  • the number of lay nozzles in the flow path 79 is preferably 2000 or less.
  • the cell culture system 200 of the tenth embodiment is a culture system used for culturing adherent cells. Although it does not specifically limit as an adherent cell, Adherent cells, such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell,
  • Adherent cells such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell
  • the culture system 200 of 10th embodiment is a pluripotent stem cell, for example, It can be used for culturing embryonic stem cells (ES cells) and inducible pluripotent stem cells (iPS cells).
  • the cell culture system 200 of the tenth embodiment includes a cell culture container 70 and a first pressing body 181 provided outside one substrate 71 of the cell culture container 70. With.
  • the cell culture system 200 of the tenth embodiment further includes first pressing body driving means 181A for driving the first pressing body 181 as shown in FIGS. 29A to 29C.
  • adherent cells are seeded and adhered to at least one of the one substrate 71 and the other substrate 72, for example, the cell adhesion surface 720 formed by the other substrate 72. It can be cultured.
  • the cells may be cultured while being adhered to the cell adhesion surface 710 of one substrate 71 and the cell adhesion surface 720 of the other substrate 72. In this case, the cultured cells are cultured on one substrate 71 and the other substrate. Although it adhere
  • the first pressing body 181 has a planar pressing surface corresponding to the planar shape of the culture chamber 75 (see FIG. 29A).
  • a portion corresponding to the pressing surface (that is, a portion corresponding to the planar shape of the culture chamber 75) can be pressed toward the culture chamber 75.
  • the planar shape of the pressing surface of the first pressing body 181 is not particularly limited as long as one substrate 71 can be pressed toward the culture chamber 75 and brought into contact with the sub-side wall 74.
  • the shape may be a rectangle or the like.
  • the pressing surface of the first pressing body 181 may be a flat surface or a curved surface as long as one substrate 71 can be pressed toward the culture chamber 75 and brought into contact with the sub-side wall 74. Good. It is preferable that the pressing surface of the first pressing body 181 has a shape that allows one substrate 71 to be in contact with a part or the whole of the end of the sub-side wall 74 on the one substrate 71 side without a gap. It is more preferable that the substrate 71 has a shape that can be brought into contact with the entire end of the sub-side wall 74 on the one substrate 71 side without a gap.
  • the pressing surface of the first pressing body 181 may have a shape capable of pressing only a portion of the one substrate 71 in contact with the sub-side wall 74, or may be in contact with the sub-side wall 74 of the one substrate 71.
  • the shape which can press also the part other than the contact part may be sufficient.
  • the whole shape of the 1st press body 181 is not specifically limited, For example, it can be set as a block shape. Therefore, in one preferable embodiment, the first pressing body 181 is a block. From the viewpoint of reducing the risk of the one substrate 71 or the other substrate 72 being broken, it is preferable that the pressing surface of the first pressing body 181 has rounded corners.
  • the material forming the pressing surface of the first pressing body 181 is preferably an elastic material. Thereby, damage of one board
  • the material forming the pressing surface of the first pressing body 181 is hard, the extent to which one substrate 71 is pushed into the culture chamber 75 (the first when the one substrate 71 is brought into contact with the sub-side wall 74)
  • the material forming the pressing surface of the first pressing body 181 is an elastic material, such highly accurate control is not required.
  • the elastic material forming the pressing surface of the first pressing body 181 include polydimethylsiloxane (PDMS).
  • the first pressing body 181 causes the first substrate When pressing 71, slippage is unlikely to occur between the first pressing body 181 and the first substrate 71, and scratches, breakage, etc. are unlikely to occur on the first substrate 71.
  • the first pressing body 181 is installed outside one substrate 71 of the cell culture container 70.
  • the desired operation of the first pressing body 181 is as follows. This can be realized by the pressing body driving means 181A.
  • the first pressing body driving means 181A drives the first pressing body 181 and the first pressing body 181 presses one substrate 71 toward the culture chamber 75 to push it into the culture chamber 75.
  • the substrate 71 can be brought into contact with the sub-side wall 74.
  • the first pressing body 181 has a pressing surface having a shape corresponding to the planar shape of the culture chamber 75 (see FIG. 29A), and a portion corresponding to the pressing surface of one substrate 71 (that is, the culture chamber 75).
  • the portion corresponding to the planar shape) can be pressed toward the culture chamber 75 and brought into contact with the sub-side wall 74.
  • a portion of the one substrate 71 that is pressed by the first pressing body can be in contact with any sub-side wall 74.
  • one substrate 71 is not pressed, as shown in FIG. 29B, one substrate 71 is separated from the sub-side wall 74, and the culture chamber 75 is an integral space.
  • the opening of the groove 78 is sealed by one substrate 71 and communicates with the inlet 76 and the outlet 77 as shown in FIG. 29C.
  • a flow path 79 is formed.
  • the plurality of culture chambers 75 may be pressed at one time by a single pressing body 181, or a plurality of pressing bodies installed corresponding to each culture chamber 75. Each culture chamber 75 may be individually pressed by 181.
  • the adherent cells are seeded and cultured on at least one of the one substrate 71 and the other substrate 72, for example, on the cell adhesion surface 720 formed by the other substrate 72.
  • a cell culture solution filled in the culture chamber 75 is used for cell culture.
  • the amount of the cell culture solution filled in the culture chamber 75 is not particularly limited, but is preferably an amount exceeding the height of the sub-side wall 74, and more preferably exceeding the height of the sub-side wall 74. The amount reaching one substrate 71 or the vicinity thereof.
  • the amount of the cell culture solution filled in the culture chamber 75 is an amount that does not exceed the height of the sub-side wall 74, the diffusion of various components in the cell culture solution is blocked by the sub-side wall 74. There is a possibility that the cell culture solution in each region in the inside may become uneven, and signal transmission between cell colonies in the culture chamber 75 may be hindered.
  • the amount of the cell culture solution filled in the culture chamber 75 is an amount exceeding the height of the sub-side wall 74, various components in the cell culture solution are transferred to the one substrate 71 and the sub-side wall 74. Since the inside of the culture chamber 75 can be diffused between the end portion on the one substrate 71 side, the cell culture solution in each region in the culture chamber 75 is unlikely to be uneven, and the inside of the culture chamber 75 Signal transmission between cell colonies is difficult to be inhibited.
  • the first pressing member 181 is driven by the first pressing member driving means 181A, and the first pressing member 181 is driven.
  • one substrate 71 is pressed toward the culture chamber 75 and brought into contact with the sub-side wall 74.
  • the one substrate 71 contacts the end of the sub-side wall 74 on the one substrate 71 side, the opening of the groove 78 is sealed, and a flow path 79 that communicates with the inlet 76 and the outlet 77 is formed. .
  • a new cell culture solution is fed from the inlet 76 to the flow path 79.
  • the new cell culture solution sent from the inlet 76 flows in one direction along the flow path 79. Therefore, the retention of the cell culture solution in the culture chamber 75, which can be caused by feeding a new cell culture solution, can be prevented, and the old cell culture solution in the culture chamber 75 can be efficiently replaced with the new cell culture solution. Can be exchanged.
  • the first pressing body 181 is driven by the first pressing body driving means 181A, and the first pressing body 181 is moved to one substrate.
  • the first pressing body 181 is released from the pressing of the one substrate 71 so as to be separated from the first pressing body 181.
  • One of the substrates 71 whose pressure has been released is separated from the sub-side wall 74 based on its stretchability.
  • a gap that is not filled with the cell culture solution is generated in the culture chamber 75. It is preferable to fill this void with a new cell culture solution.
  • the amount of the cell culture solution filled in the culture chamber 75 exceeds the height of the sub-side wall 74, and various components in the cell culture solution are on the one substrate 71 side of the one substrate 71 and the sub-side wall 74.
  • the inside of the culture chamber 75 can be diffused through the gap between the two ends. Therefore, the cell culture solution in each region in the culture chamber 75 is less likely to be uneven, and signal transmission between the cell colonies in the culture chamber 75 is less likely to be inhibited.
  • the one pressing member 181 presses the one substrate 71 while continuing to supply the new cell culture solution.
  • a new cell culture solution can be filled in the gap generated by the separation of the one substrate 71 from the sub-side wall 74.
  • the feeding of the new cell culture solution is temporarily interrupted, and then the first pressing body 181 presses one substrate 71.
  • a new cell culture solution can be filled into the gap generated by the separation of the one substrate 71 from the sub-side wall 74.
  • the cell culture solution is contained.
  • a cell culturing method comprising a step of feeding a new cell culture solution to the culture chamber 75 can be carried out. Thereby, the retention of the cell culture solution in the culture chamber 75 that may be caused by the feeding of a new cell culture solution can be prevented, and the old cell culture solution in the culture chamber 75 and the efficiency of the new cell culture solution can be reduced. Can be exchanged well.
  • the principle of the cell detachment method using the cell culture system 200 of the tenth embodiment is the same as the principle of the cell detachment method using the cell culture system of the fifth embodiment (see FIGS. 20A to 20D). It is the same.
  • cells can be peeled from the culture chamber 75 after cell culture as follows.
  • the first pressing body 181A is driven by the first pressing body driving means 181A, and as shown in FIGS. 29A and 29B, the first pressing body 181 is driven.
  • the pressing body 181 is positioned above the cell culture container 70.
  • the first pressing body driving unit 181A drives the first pressing body 181 and, as shown in FIG. 29C, presses one substrate 71 toward the culture chamber 75 by the first pressing body 181.
  • One substrate 71 is brought into contact with the sub-side wall 74.
  • the one substrate 71 contacts the end of the sub-side wall 74 on the one substrate 71 side, the opening of the groove 78 is sealed, and a flow path 79 that communicates with the inlet 76 and the outlet 77 is formed.
  • the first substrate 71 can be pressed by the first pressing body 181 while or after the cell culture solution in the culture chamber 75 is discharged. .
  • a liquid feeding means for feeding the cell recovery liquid is operated, and the cell recovery liquid is sent from the inlet 76 to the flow path 79 (see FIG. 20C).
  • the shear stress generated on the cell culture surface 720 due to the liquid feeding increases, and the cells can be detached from the cell adhesion surface 720 to collect the cells. Release of the pressure by the first pressing body 181 can be performed while feeding the cell culture solution in order to prevent a decrease in the internal pressure of the culture chamber 75.
  • the cell culture system 200 which is 10th Embodiment, a cell can be peeled from the whole cell culture surface.
  • a cell culture medium or physiological saline such as phosphate buffered saline can be used.
  • the principle of peeling cells from the cell adhesion surface using shear stress is the same as that of the cell culture system of the fifth embodiment (see FIG. 21). That is, when the diameter of the flow path 79 becomes narrower, the shear stress generated on the inner wall of the flow path 79 increases.
  • the shear stress generated on the cell culture surface of the culture chamber 75 can be increased as the height of the sub-side wall 74 is lower. Understandable. That is, by pressing one substrate 71 toward the culture chamber 75 with the first pressing body 181 and bringing it into contact with the sub-side wall 74, the cell adhesion surface corresponding to the portion pressed by the first pressing body 181. At 720, the shear stress generated by the fluid increases, which allows the cells to detach from the cell adhesion surface 720.
  • the first pressing body 181 presses the one substrate 71 toward the culture chamber 75 so as to contact the sub-side wall 74, and then the culture is performed.
  • a cell peeling method comprising a step of peeling cells by feeding a cell recovery solution to the chamber 75 can be carried out.
  • the cell culture system of the tenth embodiment can be used, for example, at the time of cell passage, for example, when the entire cell adhered to the cell culture surface is peeled and collected.
  • the cell culture system 200 is provided in a sealed container, a sealed container, a door for putting the cell culture container into and out of the sealed container, a heating unit for heating the inside of the sealed container, and a heating unit based on the temperature in the sealed container There may be further provided a temperature control unit for controlling the temperature in the sealed container by controlling the above.
  • the cell culture system 200 may also include a cell culture container mounting table.
  • the cell culture system 200 may further include a humidifying unit that humidifies the inside of the sealed container, and a humidity control unit that controls the humidity in the sealed container by controlling the humidifying unit based on the humidity in the sealed container. .
  • the cell culture system 200 includes at least one cell culture container 70.
  • the cell culture container 70 may be disposable, and the cell culture container 70 can be discarded and replaced with a new cell culture container 70 after use.
  • the cell culture container provided in the cell culture system 200 is preferably a closed cell culture container. Therefore, the cell culture system 200 is preferably a closed cell culture system.
  • the liquid feeding means for feeding the cell peeling liquid is operated, and the cell peeling liquid is supplied from the inlet 76 to the flow path 79.
  • the cell recovery solution may be supplied from the inlet 76 to the flow path 79 by operating a liquid supply means for supplying the cell recovery solution.
  • Cell adhesion can be weakened by feeding the cell detachment solution.
  • the principle of cell detachment in this modification is the same as the principle of cell detachment in the cell culture system of the seventh embodiment.
  • the cell peeling method further comprising feeding the cell peeling liquid to weaken cell adhesion may be performed before the cell recovery liquid is fed. it can. That is, according to the modification of the tenth embodiment, the step of pressing one substrate 71 toward the culture chamber 75 by the first pressing body 181 to contact the sub-side wall 74 and the cell in the culture chamber 75
  • a method for detaching cells comprising a step of feeding a detachment solution to weaken cell adhesion, and a step of detaching cells by sending a cell recovery solution to the culture chamber 75 after feeding the cell detachment solution.
  • the modified example of the tenth embodiment can be used, for example, at the time of cell passage, for example, when the whole cell adhered to the cell culture surface is peeled and collected.
  • cell detachment solutions are known as cell detachment solutions, and those skilled in the art can select an appropriate cell detachment solution as the cell detachment solution. Since the explanation regarding the cell detachment solution is the same as that of the seventh embodiment, a description thereof will be omitted.
  • the cell recovery liquid may be supplied from the inlet 76 to the flow path 79 by operating a liquid supply means for supplying the cell recovery liquid.
  • the method further includes weakening cell adhesion by applying high-frequency vibration to the cell adhesion surface of the other substrate before sending the cell recovery solution. Cell detachment methods can be implemented.
  • the step of pressing the one substrate 71 toward the culture chamber 75 by the first pressing body 181 to contact the sub-side wall 74 Implementing a cell peeling method comprising a step of applying high-frequency vibration to the cell adhesion surface 720 to weaken cell adhesion, and a step of peeling cells by feeding a cell recovery solution to the flow path 79.
  • the modified example of the tenth embodiment can be used, for example, at the time of cell passage, for example, when the whole cell adhered to the cell culture surface is peeled and recovered.
  • Ultrasonic waves can be used as the high-frequency vibration.
  • a piezoelectric element a plastic ultrasonic welder, a commercially available ultrasonic generator for cell disruption, or an ultrasonic generator for an ultrasonic cleaner is used. It can be generated by using as a high frequency vibration source.
  • the high frequency is, for example, preferably 20 Hz to 10 GHz, more preferably 16 kHz to 10 GHz, further preferably 16 kHz to 1 GHz, and further preferably 20 kHz to 100 kHz.
  • the high frequency vibration of 70 kHz can be concentrated on a certain circular portion (for example, ⁇ 2 mm) to give the high frequency vibration.
  • the cell culture system 200 of the tenth embodiment may further include a high frequency vibration generating unit that applies high frequency vibration to the cell adhesion surface 720.
  • the high frequency vibration generating unit can be configured in the same manner as the high frequency vibration generating unit 19 shown in FIGS. 22A to 22F, and the description regarding the high frequency vibration generating unit 19 can be applied.
  • the high-frequency vibration generating unit can be installed outside the other substrate 72 of the cell culture system 200 of the tenth embodiment.
  • the high frequency vibration generating unit has a surface having a shape corresponding to the planar shape of the culture chamber 75, and can apply high frequency vibration to the entire cell adhesion surface 720.
  • the high-frequency vibration generating unit has a surface smaller than the planar shape of the culture chamber 75, and the cell adhesion of the cell adhesion surface 720 can be weakened in a region-specific manner by high-frequency vibration.
  • the cell peeling method before the cell recovery solution is sent, further includes applying high-frequency vibration to the cell adhesion surface of the other substrate 72 to weaken the cell adhesion. Can do.
  • the high-frequency vibration is pressed against one substrate 71 by the first pressing body 181 after pressing the one substrate 72 toward the culture chamber 75 to contact the sub-side wall 74 or by the first pressing body 181. It can be applied to one substrate 71 or the other substrate 72 before it is done.
  • ultrasonic waves can be used as the high-frequency vibrations.
  • the ultrasonic waves are, for example, piezoelectric elements, plastic ultrasonic welders, commercially available ultrasonic waves for cell disruption. It can generate
  • the high frequency is, for example, preferably 20 Hz to 10 GHz, more preferably 16 kHz to 10 GHz, further preferably 16 kHz to 1 GHz, and further preferably 20 kHz to 100 kHz.
  • the high frequency vibration of 70 kHz can be concentrated on a certain circular portion (for example, ⁇ 2 mm) to give the high frequency vibration.
  • High-frequency vibration can peel cells when a high-frequency vibration source is brought close to or in contact with one substrate 71 or the other substrate 72 having a cell adhesion surface from the outside of the culture vessel. Cells that have been detached from the cell adhesion surface by applying high-frequency vibration can be cultured well thereafter.
  • High frequency vibration can also be used to weaken the adhesion between cells and the cell adhesion surface.
  • the cell adhesion surface is preferably formed of a stretchable material in order to reduce the risk of breakage due to friction and heat generated by ultrasonic vibration.
  • a single high-frequency vibration generator may apply high-frequency vibrations to the plurality of culture chambers 75, or each culture chamber 75 may be individually subjected to high-frequency vibrations. May be given.
  • the eleventh embodiment will be described below.
  • the cell culture container in the eleventh embodiment is the same as the cell culture container 70 in the tenth embodiment.
  • the cell culture system 300 of the eleventh embodiment is a culture system used for culturing adherent cells. Although it does not specifically limit as an adherent cell, Adhesive cells, such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell, are mentioned,
  • Adhesive cells such as a pluripotent stem cell, a stem cell, a progenitor cell, a somatic cell, and a germ cell
  • the culture system 300 of 11th embodiment is a pluripotent stem cell, for example, It can be used for culturing embryonic stem cells (ES cells) and inducible pluripotent stem cells (iPS cells).
  • the eleventh cultured cell system 300 includes a cell culture container 70, a first pressing body 181 provided outside one substrate 71 of the cell culture container 70, and a first press body 181.
  • the cell culture system 300 of the eleventh embodiment drives the first pressing body driving means 181A for driving the first pressing body 181 and the second pressing body 182. And a second pressing body driving means 182A.
  • the cell culture system 300 according to the eleventh embodiment is different from the cell culture system 200 according to the tenth embodiment in that a second pressing body 182 is provided. That is, the cell culture system 300 according to the eleventh embodiment is different from the cell culture system 200 according to the tenth embodiment in that the second pressing body 182 is used when cells are peeled.
  • adherent cells are seeded and adhered to at least one of the one substrate 71 and the other substrate 72, for example, the cell adhesion surface 720 formed by the other substrate 72. It can be cultured.
  • the cells may be cultured while being adhered to the cell adhesion surface 710 of one substrate 71 and the cell adhesion surface 720 of the other substrate 72. In this case, the cultured cells are cultured on one substrate 71 and the other substrate. Although it adhere
  • the first pressing body 181 is the same as the cell culture system 200 of the tenth embodiment.
  • the second pressing body 182 has a pressing surface that can enter the groove 78, and presses one substrate 71 toward the culture chamber 75 to press the groove 78. Can be pushed in.
  • the second pressing body 182 has a pressing surface smaller than the planar shape of the culture chamber 75 (see FIG. 30A). (That is, a part of the portion corresponding to the planar shape of the groove portion 78) can be pressed. Thereby, only the cells adhered to the pressed portion of one substrate 71 (or the portion corresponding to the pressed portion in the other substrate 72) can be peeled in a region-specific manner.
  • the shape of the pressing surface of the second pressing body 182 is not particularly limited, and examples thereof include a circle and a rectangle.
  • the pressing surface of the second pressing member 182 in a cell culture system for culturing pluripotent stem cells, a circular diameter of about 0.4mm ⁇ 3.4mm (0.12mm 2 ⁇ 9mm 2 approximately in the area) It can be set as a pressing surface.
  • cells can be exfoliated in a region-selective manner, for example, each colony formed by pluripotent stem cells during adhesion culture can be selectively exfoliated.
  • the pressing surface of the second pressing body 182 is rectangular, it is easier to increase the shear stress than when the pressing surface is circular.
  • the pressing surface of the second pressing member 182 and the rectangular pressing surface of about one side 0.35mm ⁇ 3mm (0.12mm 2 ⁇ 9mm 2 in area) for example, be a pressing surface of the square .
  • the pressing surface of the second pressing body 182 may have a planar shape or a curved surface shape, for example, a spherical shape.
  • the whole shape of the 2nd press body 182 is not specifically limited, For example, it can be set as a block shape. Therefore, in one preferable embodiment, the second pressing body 182 is a block. From the viewpoint of reducing the risk of breaking one substrate 71 or the other substrate 72, it is preferable that the pressing surface of the second pressing body 182 has rounded corners.
  • the material forming the pressing surface of the second pressing body 182 is preferably an elastic material. Thereby, damage of one board
  • the material forming the pressing surface of the second pressing body 182 is hard, the degree to which one substrate 71 is pushed into the culture chamber 75 (the second pressing when the one substrate 71 is pushed into the groove 78). With respect to the position of the body 182, high-precision control is required, but when the material forming the pressing surface of the second pressing body 182 is an elastic material, such high-precision control is not required.
  • Examples of the elastic material forming the pressing surface of the second pressing body 182 include polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • silicone rubber is used as the elastic material for forming the first substrate 71
  • the second pressing body 182 causes the first substrate When pressing 71, slippage is unlikely to occur between the second pressing body 182 and the first substrate 71, and scratches, breakage, etc. are unlikely to occur on the first substrate 71.
  • the second pressing body 182 is installed outside one substrate 71 of the cell culture container 70, and the desired operation of the second pressing body 182 is as follows. This can be realized by the pressing body driving means 182A.
  • the second pressing body 182A is driven by the second pressing body driving means 182A, and as shown in FIG. 30C, one substrate 71 is pressed toward the culture chamber 75 by the second pressing body 182 to form the groove portion. 78 can be pushed into.
  • the second pressing body driving means 182A can drive the second pressing body 182 and scrape off the cells adhered to the cell adhesion surface with the one substrate 71.
  • the second pressing body driving means 182A drives the second pressing body 182 so that the cells are sandwiched between the one substrate 71 and the other substrate 72 and are scraped off.
  • the second pressing body driving unit 182A is not particularly limited.
  • the second pressing body 182 is placed on the cell adhesion surface 720 of the other substrate 72 while the one substrate 71 and the other substrate 72 are in contact with each other. (See FIGS. 19A to 19D).
  • the second pressing body 182 may be driven by a simple linear motion, or may be accompanied by a motion such as a rotational motion or a reciprocating motion.
  • the cell culture system of the present embodiment includes the second pressing body driving unit 182A that drives the second pressing body 182 to scrape off the cells adhered to the cell adhesion surface with the one substrate 71. Further, when the cells adhered to the cell adhesion surface are scraped off by one substrate 71, the second pressing body 182 can be fixed and the culture vessel 70 can be driven.
  • the principle of the cell detachment method using the cell culture system 300 of the eleventh embodiment is the same as the principle of the cell detachment method using the cell culture system of the fourth embodiment (see FIGS. 19A to 19D). It is the same.
  • the cells can be peeled from the culture chamber 75 using the second pressing body 182 after cell culture as follows. .
  • the second pressing body 182A is driven by the second pressing body driving means 182A, and as shown in FIGS. 30A and 30B, the second pressing body 182A is driven.
  • the pressing body 182 is positioned above the cell culture container 70.
  • the second pressing body 182A is driven by the second pressing body driving means 182A, and one substrate 71 is pressed toward the culture chamber 75 by the second pressing body 182 as shown in FIG. 30C. Push into the groove 78.
  • the volume of the culture chamber 75 varies, but the increase of the internal pressure in the culture chamber 75 can be suppressed by the other substrate 72 not pressed by the second pressing body 182 expanding outward.
  • the pressing of the one substrate 71 by the second pressing body 182 may be performed while discharging the cell culture solution in the culture chamber 75 or after discharging it.
  • the second pressing body driving means 182A drives the second pressing body 182 to scrape off the cells through one substrate 71 (see FIG. 19C).
  • the second pressing body driving means 182A may be operated while feeding the cell recovery liquid to the culture chamber 75.
  • the second pressing body 182 is driven by the pressing body driving means 182A, and the pressing of the first substrate 71 by the second pressing body 182 is released.
  • the first pressing body 181 is driven by the first pressing body driving means 181A, and the first pressing body 181 is positioned above the cell culture container 70 (see FIG. 29B).
  • the first pressing body driving means 181A drives the first pressing body 181 and the first pressing body 181 presses one substrate 71 toward the culture chamber 75 to contact the sub-side wall 74. (See FIG. 29C). Accordingly, the opening of the groove 78 is sealed by the one substrate 71, and a flow path 79 communicating with the inlet 76 and the outlet 77 is formed.
  • the cell recovery solution is sent from the inlet 76 to the flow path 79, and the scraped cells are recovered or removed from the outlet 77.
  • cells can be detached selectively in a region. That is, according to the cell culture system of the eleventh embodiment, the second pressing body 182 presses the one substrate 71 toward the culture chamber 75 and presses it into the groove portion 78, and the second pressing body driving means.
  • a cell peeling method comprising a step of peeling cells by driving 182A and scraping the cells with one substrate 71 can be performed.
  • the cell culture system 300 according to the eleventh embodiment can be used when a specific cell, for example, a defective pluripotent stem cell colony is detached and removed from the cell adhesion surface.
  • the cell culture system 300 of the eleventh embodiment can be used when recovering and substituting specific cells from the cell adhesion surface.
  • the cell culture system 300 of the eleventh embodiment includes a sealed container, a door provided in the sealed container, a door for taking the cell culture container into and out of the sealed container, a heating unit for heating the sealed container, and a sealed container You may further provide the temperature control part which controls the temperature in an airtight container by controlling a heating part based on this temperature.
  • the cell culture system 300 of the eleventh embodiment may also include a mounting table for cell culture containers.
  • the eleventh cell culture system 300 further includes a humidifying unit for humidifying the inside of the sealed container, and a humidity control unit for controlling the humidity in the sealed container by controlling the humidifying unit based on the humidity in the sealed container. It may be.
  • the cell culture system 300 includes at least one cell culture container 70.
  • the cell culture container 70 may be disposable, and the cell culture container 70 can be discarded and replaced with a new cell culture container 70 after use.
  • the cell culture container 70 provided in the cell culture system 300 of the eleventh embodiment is preferably a closed cell culture container. Therefore, the cell culture system 300 of the eleventh embodiment is preferably a closed cell culture system.
  • the second pressing body 182 presses one substrate 71 toward the culture chamber 75 and pushes it into the groove 78.
  • the second pressing body driving means 182A drives the second pressing body 182 to generate high-frequency vibration from the second pressing body 182 and then the second pressing body driving means 182A is driven to drive one substrate.
  • the cells may be scraped off by 71.
  • the high-frequency vibration is transmitted to the cell adhesion surface 720 and the adhesion between the cell adhesion surface 720 and the cell can be weakened, so that the cells are easily scraped off.
  • the second pressing body 182 presses one substrate 71 toward the culture chamber 75 and pushes it into the groove portion 78.
  • the second pressing body driving means 182A drives the second pressing body 182 to generate high-frequency vibration from the second pressing body 182 and then the second pressing body driving means 182A is driven to drive one substrate.
  • 71 can be performed, further comprising scraping the cells with 71.
  • the cells that are exfoliated in a region-specific manner may be cells for recovery and passage, or cells for removal (for example, defective pluripotent stem cell colonies).
  • Example 1 Examination of human iPS cells into single cells and subsequent passage methods When pluripotent stem cells such as ES cells and iPS cells are separated into single cells during passage, cell death is induced. Is done. In addition, there is a concern that cells may start to differentiate when embryoid bodies are formed. In this example, it was examined whether cell death or differentiation problems would occur when cell clumps were rapidly formed after being dissociated into single cells and then passaged.
  • human iPS cells established strain by Kawasada Laboratory, Cell Evaluation Group, Advanced Medical Promotion Foundation
  • the culture was performed under feeder-less conditions.
  • a medium obtained by adding bFGF (manufactured by Wako Pure Chemical Industries, product number: 064-04541) final concentration of 5 ng / mL to ReproFF2 medium (manufactured by ReproCell, product number: RCHEMD006) was used.
  • a culture vessel a 10 mm cell culture dish (manufactured by BD, product number: REF353003) is used, and in order to ensure adhesion of iPS cells to the culture vessel, according to the method described in the manufacturer's instruction manual, Before culturing, the inside of the culture vessel was coated with ECM.
  • ECM BD Matrigel (manufactured by BD, product number: 356234) was used.
  • the cells were cultured until they became confluent by a conventional method. Thereafter, the medium was removed by aspiration for passage, and the cells were washed once with 10 mL of phosphate buffered saline (Life Technologies, product number: 14190). Thereafter, the cells were treated with 1 mL of accutase solution (Innovative cell technologies, product number: AT104) at 37 ° C. for 5 minutes, and the cells were collected in a tube.
  • phosphate buffered saline Life Technologies, product number: 14190
  • the cells were collected by centrifugation (440 g, 5 minutes), the supernatant was removed by aspiration, the cells were resuspended in 1 mL of medium, and ROCK inhibitor Y-27632 (manufactured by STEMGENT, product number: 04-0012) was added to the medium. ) was added to a final concentration of 10 ⁇ M to obtain a cell suspension. The cells were then crushed by pipetting water flow until single cell level. The cell concentration in the cell suspension was adjusted, and the cell suspension was injected into the wells of Aggrewell 800 (manufactured by STEMCELL Technologies). Thereafter, the AggreWell 800 was centrifuged (2000 g, 5 minutes) or not, and cultured at 37 ° C.
  • FIG. 11A The state of the cells immediately after seeding and after 1 day when the centrifugation was performed and when the centrifugation was not performed was as shown in FIG. 11A. That is, when centrifugation was performed, cells were collected at the bottom of the inverted pyramid-shaped well immediately after seeding (FIG. 11A—upper left), but when centrifugation was not performed, cells were not collected ( FIG. 11A-upper right). However, after one day, the cells gathered at the bottom of the well to form a cell conglomerate whether centrifugation was performed (FIG. 11A—lower left) or not (FIG. 11A—lower right). Moreover, the obtained cell clump had a substantially uniform size (FIG. 11B). In addition, cell agglomeration was formed at 8 hours and 12 hours, respectively, in the wells of AggreWell, and 8 to 12 hours was sufficient (data not shown).
  • the obtained cell clumps were collected with a pipette so as not to break as much as possible, and seeded on a 6-well plate (300 cell clumps / well).
  • a pipette so as not to break as much as possible
  • a 6-well plate 300 cell clumps / well.
  • the cell conglomerate obtained in this example has a three-dimensional structure artificially accumulated, the situation is greatly different from that of normal subculture, but in this example, it was unexpectedly produced in this way. Even when a multi-layered cell agglomeration was cultured, it naturally developed, and after that, it was possible to form a colony of pluripotent stem cells found in normal culture and to carry out the culture suitably.
  • cell growth after seeding in a 6-well plate was compared using a growth curve, but there was no significant difference in the cell growth rate (FIG. 13).
  • the cells are seeded on a 6-well plate, collected 5 days later, fixed with paraformaldehyde, and then subjected to fluorescent immunostaining. It was. Nuclear staining was performed by incubating the cells for 15 minutes in phosphate buffered saline containing 1 ⁇ g / mL DAPI. Fluorescent immunostaining uses an anti-Nanog antibody (ReproCell, product number: RAB0003P) as an antibody to confirm Nanog expression. Oct3 / 4 is an anti-Oct3 / 4 antibody (Santa Cruz Biotechnology, product number) : Sc-5279) according to a conventional method. The bright field image was acquired using a phase contrast microscope (Olympus, product number: IX-81).
  • Example 2 Examination of seeding method of cell agglomeration on culture surface of culture vessel
  • the cell agglomerate formed in the microwell was passaged to a new culture vessel by collecting with a pipette. Careful pipetting operation was required, taking time to avoid breaking the cell conglomerate. Therefore, in this example, a simpler cell agglomeration method was examined.
  • the inventors examined the use of a closed culture vessel having a surface having a microwell and a culture surface, and these two surfaces are arranged to face each other.
  • the surface of the closed culture vessel having the microwells was formed by arranging square-shaped microwells having a square bottom shape and an opening of 1000 ⁇ m ⁇ 1000 ⁇ m in a grid shape.
  • the culture surface was coated with BD Matrigel (trademark).
  • FIG. 15 is a diagram showing the arrangement of cell clumps dropped on the culture surface. As shown in FIG. 15, the cell clumps were regularly aligned on the culture surface.
  • the arrangement of these cell agglomerates reflects the arrangement pattern of the used microwells, and the interval between the cell agglomerates in FIG. 15 is the pitch (1000 ⁇ m) of the microwells of the closed culture vessel used in this example. Matched.
  • the cell agglomeration in the microwell can be dropped onto the culture surface of the culture vessel while maintaining the arrangement pattern of the microwell. It is considered that the seeding position of the cell cluster can be freely controlled by changing the arrangement of the microwells. In addition, it became clear that the sowing and the sowing position can be controlled by a very easy operation of turning the container upside down.
  • Example 1 even when pluripotent stem cells are dispersed into single cells, cell clumps are formed immediately thereafter, so that it can be well cultured while maintaining an undifferentiated state. there were. Further, by seeding the cell suspension in a culture vessel having a surface on which a plurality of microwells are arranged, it was possible to easily form a cell aggregate having a uniform size. Furthermore, the cell clumps developed rapidly after seeding in the culture vessel, and the cells proliferated well. Since the size of the formed cell clump was uniform, the cell clump development rate and the subsequent growth rate were also uniform. Further, according to Example 2, the cell clumps in the microwells could be seeded on the culture surface of the culture vessel by a simple operation.
  • the arrangement pattern of the seeded cell agglomeration reflects the arrangement pattern of the microwells, and it was shown that the cell agglomeration can be precisely seeded by a simple operation.
  • formation of a uniform cell clump, uniform seeding of the cell clump, and the like can be performed by a very simple mechanical operation.
  • the method of the present invention not only facilitates maintaining the quality of pluripotent stem cells, but also opens the way to full automation of subculture of pluripotent stem cells.
  • Example 3 Production of Cell Culture Container
  • a culture container (see FIGS. 25A to 25F) was prototyped.
  • the cell culture container produced in this example was produced using the plan view shown in FIG. 25A, the front view shown in FIG. 25B, the side view shown in FIG. 25C, and the bottom view shown in FIG.
  • a cross-sectional view taken along the line AA of this cell culture container is shown in FIG. 25E, and a cross-sectional view taken along the line BB is shown in FIG. 25F.
  • the cell culture vessel frame (hard frame with flow channel grooves) was made by machining polycarbonate. Two thin films of PDMS (polydimethylsiloxane) were attached to the frame with a silicone adhesive (Cemedine Super X), one on each of the upper and lower surfaces (FIGS. 25A to 25E).
  • PDMS polydimethylsiloxane
  • the PDMS thin film was produced by a spin coating method. Specifically, first, 10 mL of SILPOT 184 manufactured by Toray Dow Corning was dropped onto a mirror silicon (Si) wafer substrate, and vacuum defoaming was performed. Subsequently, it was spread on a Si wafer under a spin coating condition of 1000 rpm / 30 seconds. Thereafter, the PDMS thin film was peeled off from the Si wafer after heat treatment at 120 ° C./2 hours in an air atmosphere.
  • SILPOT 184 manufactured by Toray Dow Corning
  • a transparent PDMS thin film having a thickness of 0.065 mm could be produced under the above conditions.
  • a rubber plug made of isopropylene was detachably installed at the entrance of the container.
  • the dimensions of the flow path were 1 mm wide and 1 mm deep at the bent portion connecting the culture chambers, and the culture chamber was 5 mm wide and 3 mm deep.
  • the taper portion connecting the bent portion and the culture chamber had a shape spreading at an apex angle of 60 °, and the depth was 3 mm.
  • the overall dimensions of the container were 127.6 mm ⁇ 85.4 mm ⁇ 10 mm (H) (excluding the rubber stopper).
  • Example 4 Pressing with a pressing body and peeling of cells by enzyme treatment
  • a cell peeling experiment was conducted using the cell culture container prepared in Example 3 and a block-shaped pressing body.
  • iPS cells were prepared as follows. First, iPS cells were seeded in the cell culture vessel prepared in Example 3, and adherently cultured on PDMS by a normal iPS cell culture protocol (see, for example, FIG. 26A). An exfoliation experiment was performed on an iPS cell colony having a diameter of about 2 mm.
  • the peeling experiment was performed as follows. First, with the pressing body 18 approaching the other substrate 20 to a distance of 1.0 mm while removing the medium, the collagenase IV solution (collagenase IV manufactured by Invitrogen Corporation was adjusted to a concentration of 100 U / mL to establish a Hanks equilibrium. The cell detachment solution adjusted to a concentration of 1.25 mg / mL trypsin in a salt solution (solution dissolved in HBSS solution) was supplied at 2 mL / min to exchange the medium with the cell detachment solution (for example, FIG. 20B). . Next, cell detachment liquid treatment was performed at 37 ° C. for 10 minutes.
  • FIG. 26A shows a phase contrast microscopic image of the cells immediately after the culture
  • FIG. 26B shows a phase contrast microscopic image of the cells after treatment with the cell detachment solution
  • FIG. 26C shows a state after collecting the cells by liquid feeding. The phase-contrast microscope image of the cell adhesion surface of is shown.
  • the present invention it is possible to reduce the amount of the cell detachment solution and the medium to be used, and to apply a desired shear stress, and to detach and collect cells with high efficiency in closed culture.
  • the shear stress generated when the liquid is fed into the culture chamber is pressed by the pressing surface. Increase in area. And when this shear force was utilized, the cell was able to be peeled from the cell adhesion surface corresponding to the area
  • a pressing body having a pressing surface corresponding to the planar shape of the entire culture chamber was used, cells could be detached from the cell adhesion surface corresponding to the planar shape of the culture chamber.
  • a cell detachment solution containing an enzyme for detaching cells may be used, but since the volume of the culture chamber can be reduced by pressing, an expensive enzyme The amount used could be reduced.

Landscapes

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

Abstract

La présente invention a pour objet un récipient de culture de cellules pour la sous-culture de façon égale et efficace de cellules, en particulier de cellules souches pluripotentes, un procédé de sous-culture de cellules utilisant ledit récipient de culture de cellules et un système de sous-culture de cellules qui utilise ledit récipient de culture. Le récipient de culture de cellules selon l'invention est pourvu d'une première plaque de base, d'une seconde plaque de passe disposée à l'opposé de la première plaque de base et d'une paroi latérale qui est interposée entre la première plaque de base et la seconde plaque de base et qui forme un trajet d'écoulement et une chambre de culture qui a une surface de fixation de cellules formée par la première plaque de base et/ou la seconde plaque de base. La première plaque de base est constituée d'une matière étirable.
PCT/JP2014/084597 2014-01-09 2014-12-26 Récipient de culture de cellules, système de sous-culture de cellules et procédé de sous-culture de cellules WO2015105029A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2014002626A JP2017046591A (ja) 2014-01-09 2014-01-09 細胞培養システムおよび細胞の剥離方法
JP2014-002626 2014-01-09
JP2014002636A JP2017046592A (ja) 2014-01-09 2014-01-09 細胞培養容器および細胞継代培養システム並びに細胞継代培養方法
JP2014-002636 2014-01-09

Publications (1)

Publication Number Publication Date
WO2015105029A1 true WO2015105029A1 (fr) 2015-07-16

Family

ID=53523862

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/084597 WO2015105029A1 (fr) 2014-01-09 2014-12-26 Récipient de culture de cellules, système de sous-culture de cellules et procédé de sous-culture de cellules

Country Status (1)

Country Link
WO (1) WO2015105029A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017006942A1 (fr) * 2015-07-06 2017-01-12 株式会社朝日ラバー Récipient renfermant des cellules et procédé de culture cellulaire à l'aide d'un tel récipient
WO2017111054A1 (fr) * 2015-12-25 2017-06-29 東京エレクトロン株式会社 Récipient de culture
WO2019069931A1 (fr) * 2017-10-03 2019-04-11 公立大学法人大阪府立大学 Récipient de culture cellulaire, procédé d'acquisition de cellules et procédé de culture cellulaire
JP2019126326A (ja) * 2018-01-26 2019-08-01 東ソー株式会社 多能性幹細胞の剥離方法
CN110468049A (zh) * 2018-05-10 2019-11-19 澳门大学 用于制备干细胞球的装置、制备干细胞球的方法以及保存干细胞的方法
CN111511898A (zh) * 2018-01-09 2020-08-07 东洋制罐集团控股株式会社 细胞培养方法及装置
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

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02501529A (ja) * 1987-05-04 1990-05-31 ベインズ，アルバート　ジェー． 細胞培養装置用の生体適合性ポリオルガノシロキサン組成物
JP2006314204A (ja) * 2005-05-10 2006-11-24 Chiyoda Corp 培養容器からの動物細胞の剥離方法および剥離回収方法
JP2006325537A (ja) * 2005-05-30 2006-12-07 Yamaha Corp 細胞培養チップおよびその製造方法
JP2008504845A (ja) * 2004-06-07 2008-02-21 バイオプロセッサーズ コーポレイション リアクター環境条件の制御
JP2008054521A (ja) * 2006-08-29 2008-03-13 Canon Inc 細胞培養処理装置及び細胞培養処理方法
JP2010151717A (ja) * 2008-12-26 2010-07-08 Aida Eng Ltd マイクロ流路チップ
JP2010233456A (ja) * 2009-03-30 2010-10-21 Kitakyushu Foundation For The Advancement Of Industry Science & Technology 細胞集合体形成器具、細胞集合体培養器具、細胞集合体転写キット及び細胞集合体の培養方法
JP2011036221A (ja) * 2009-08-18 2011-02-24 Stem Biomethod Corp 細胞収容装置
JP2012175983A (ja) * 2012-06-19 2012-09-13 Dainippon Printing Co Ltd 細胞培養用基板

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02501529A (ja) * 1987-05-04 1990-05-31 ベインズ，アルバート　ジェー． 細胞培養装置用の生体適合性ポリオルガノシロキサン組成物
JP2008504845A (ja) * 2004-06-07 2008-02-21 バイオプロセッサーズ コーポレイション リアクター環境条件の制御
JP2006314204A (ja) * 2005-05-10 2006-11-24 Chiyoda Corp 培養容器からの動物細胞の剥離方法および剥離回収方法
JP2006325537A (ja) * 2005-05-30 2006-12-07 Yamaha Corp 細胞培養チップおよびその製造方法
JP2008054521A (ja) * 2006-08-29 2008-03-13 Canon Inc 細胞培養処理装置及び細胞培養処理方法
JP2010151717A (ja) * 2008-12-26 2010-07-08 Aida Eng Ltd マイクロ流路チップ
JP2010233456A (ja) * 2009-03-30 2010-10-21 Kitakyushu Foundation For The Advancement Of Industry Science & Technology 細胞集合体形成器具、細胞集合体培養器具、細胞集合体転写キット及び細胞集合体の培養方法
JP2011036221A (ja) * 2009-08-18 2011-02-24 Stem Biomethod Corp 細胞収容装置
JP2012175983A (ja) * 2012-06-19 2012-09-13 Dainippon Printing Co Ltd 細胞培養用基板

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, N. ET AL.: "Microfluidic chips controlled with elastomeric microvalve arrays.", J. VIS. EXP., vol. 8, no. 296, 2007, pages 1 - 3 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017006942A1 (fr) * 2015-07-06 2017-01-12 株式会社朝日ラバー Récipient renfermant des cellules et procédé de culture cellulaire à l'aide d'un tel récipient
US10563159B2 (en) 2015-07-06 2020-02-18 Asahi Rubber Inc. Cell-holding container and cell culture method using same
WO2017111054A1 (fr) * 2015-12-25 2017-06-29 東京エレクトロン株式会社 Récipient de culture
WO2019069931A1 (fr) * 2017-10-03 2019-04-11 公立大学法人大阪府立大学 Récipient de culture cellulaire, procédé d'acquisition de cellules et procédé de culture cellulaire
JP7489054B2 (ja) 2017-10-03 2024-05-23 公立大学法人大阪 細胞の取得方法、および細胞の培養方法
US20200318053A1 (en) * 2017-10-03 2020-10-08 University Public Corporation Osaka Cell culture container, method for acquiring cells, and method for culturing cells
JPWO2019069931A1 (ja) * 2017-10-03 2020-10-22 公立大学法人大阪 細胞培養容器、細胞の取得方法、および細胞の培養方法
CN111511898B (zh) * 2018-01-09 2024-01-02 东洋制罐集团控股株式会社 细胞培养方法及装置
CN111511898A (zh) * 2018-01-09 2020-08-07 东洋制罐集团控股株式会社 细胞培养方法及装置
JP2019126326A (ja) * 2018-01-26 2019-08-01 東ソー株式会社 多能性幹細胞の剥離方法
CN110468049A (zh) * 2018-05-10 2019-11-19 澳门大学 用于制备干细胞球的装置、制备干细胞球的方法以及保存干细胞的方法
JP7352982B2 (ja) 2019-07-25 2023-09-29 株式会社サンプラテック 培養液収容容器
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

Similar Documents

Publication Publication Date Title
WO2015105029A1 (fr) Récipient de culture de cellules, système de sous-culture de cellules et procédé de sous-culture de cellules
US8114646B2 (en) Method for ultrasonic cell removal
JP6227846B2 (ja) 細胞保持容器及びそれを用いた細胞培養方法
US20100068793A1 (en) Devices and methods for production of cell aggregates
US20100184182A1 (en) Method for preparing biological tissue
KR101828910B1 (ko) 세포 박리 방법
WO2019138956A1 (fr) Procédé et dispositif de culture cellulaire
US20100184222A1 (en) Method for preparing biological tissue
WO2014017481A1 (fr) PROCÉDÉ DE DÉTACHEMENT DE CELLULES PAR CHOIX DE RÉGION ET PROCÉDÉ DE CULTURE AINSI QUE PROCÉDÉ DE REPIQUAGE DE CELLULES METTANT EN œUVRE CE PROCÉDÉ DE DÉTACHEMENT
EP2949746A1 (fr) Procédé de sous-culture de cellules souches pluripotentes
JP2006314204A (ja) 培養容器からの動物細胞の剥離方法および剥離回収方法
US10351811B2 (en) Cell culture container
JP2005278564A (ja) 細胞培養装置
JP2017046592A (ja) 細胞培養容器および細胞継代培養システム並びに細胞継代培養方法
EP2230014A1 (fr) Dispositifs et procédés pour la production d'agrégats cellulaires
WO2020196635A1 (fr) Dispositif de manipulation de cellules et procédé de manipulation de cellules
JP7016371B2 (ja) 接着細胞培養基質サンプリングデバイス
Kim et al. Microfabricated embryonic stem cell divider for large-scale propagation of human embryonic stem cells
JP2005295969A (ja) 細胞分離装置
JP2017046591A (ja) 細胞培養システムおよび細胞の剥離方法
JP2023135601A (ja) 細胞の回収方法および細胞回収装置
JP2004105139A (ja) 細胞の遠心分離方法及びその遠心分離装置
JP2016000008A (ja) 接着細胞の培養装置および培養方法
Choi et al. Induction of Embryoid Body formation using a concave PDMS mold
CA2659133A1 (fr) Dispositif et methodes de production d'agregats cellulaires

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: 14877645

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: 14877645

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP