WO2020116254A1 - Dispositif de culture cellulaire - Google Patents

Dispositif de culture cellulaire Download PDF

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Publication number
WO2020116254A1
WO2020116254A1 PCT/JP2019/046230 JP2019046230W WO2020116254A1 WO 2020116254 A1 WO2020116254 A1 WO 2020116254A1 JP 2019046230 W JP2019046230 W JP 2019046230W WO 2020116254 A1 WO2020116254 A1 WO 2020116254A1
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membrane
microchannel
porous
cell culture
porous membrane
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PCT/JP2019/046230
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English (en)
Japanese (ja)
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晃寿 伊藤
圭介 奥
孝浩 大場
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富士フイルム株式会社
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Publication of WO2020116254A1 publication Critical patent/WO2020116254A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present disclosure relates to a cell culture device.
  • a cell culture device having a flow channel of a micrometer order width called a micro flow channel defined by a plurality of cavity members is known (for example, Japanese Patent Nos. 5700460 and 5771962).
  • the microchannel is divided into upper and lower parts by a porous membrane, and the microchannel is deformed by pressure from both sides of the porous membrane.
  • Cell culture devices that expand and contract a porous membrane by means of this are also known.
  • cell culture devices that imitate biological functions by incorporating a porous membrane in the microchannel and culturing cells on one or both sides of this porous membrane have attracted attention as a drug discovery support tool.
  • cell culture using a cell culture device it is possible to control the orientation of cells by applying mechanical stress, in addition to supplying nutrients by perfusion of a medium.
  • the cell culture device is expanded and contracted with the deformation of the microchannel. It is also possible to repeatedly apply mechanical stress to cell tissues and promote orientation control and maturation of cells.
  • the extension characteristics of the porous membrane are much lower than the environment in which the target cells are supported in vivo (eg, blood vessel wall, intestinal wall). Therefore, there is a problem in that it is difficult to give sufficient deformation to the porous membrane to apply mechanical stress to the cells. Moreover, in order to give sufficient deformation to the porous membrane, it is necessary to apply a corresponding force, but the elongation property of the porous membrane may not be able to withstand such force and may cause damage to the cell culture device. ..
  • the present disclosure easily reproduces the mechanical stress applied to cells in vivo by using a porous membrane that can be stretched with a force smaller than the force required for stretching the porous membrane used in the prior art.
  • An object is to provide a possible cell culture device.
  • a cell culture device is arranged between a pair of cavity members facing each other and having microcavities formed on the facing surfaces, and the facing surfaces of the pair of cavity members, and A porous film having a plurality of openings arranged in a honeycomb shape on one side or both sides of the porous film, which are connected to the cavity member or the porous film, and apply at least a force along the thickness direction to the porous film so that the porous film has at least the thickness.
  • a membrane stretching mechanism capable of stretching along a direction, wherein the porous membrane has a plurality of intramembrane spaces communicating in the thickness direction from each of the plurality of openings, and adjacent membrane spaces are porous.
  • a porous film at least along a thickness direction means extending up or down along a thickness direction.
  • the microcavities are separated by the porous film.
  • the porous membrane has openings arranged in a honeycomb shape on one side or both sides, has an intramembrane space communicating from the openings in the thickness direction, and adjacent intramembrane spaces communicate with each other inside the porous membrane. It has a horizontal communication structure. Furthermore, the average opening diameter of the openings is 1 ⁇ m or more and 200 ⁇ m or less, the porosity of the porous film is 40% or more and 90% or less, the film thickness of the porous film is 0.5 ⁇ m or more and 100 ⁇ m or less, and 20% of the porous film.
  • the tensile force per unit width required for stretching is 0.1 N/m or more and 20 N/m or less.
  • a cell culture device is the cell culture device according to the first aspect, wherein the porous membrane has a plurality of openings on both sides, and the opening on one side is the opening on the other side through the intramembrane space. Is in communication with.
  • the intra-membrane space penetrates in the thickness direction of the porous film from the opening on one surface to the opening on the other surface, it is between the pair of micro cavities separated by the porous film. Liquids and substances can be circulated.
  • the cell culture device is the cell culture device according to the first or second aspect, wherein the other of the microcavities is a well having an opening on the side opposite to the porous membrane.
  • one of the pair of microcavities is a microchannel, and the other is a well having an opening on the side opposite to the porous membrane, and thus seeding cells into the well through this opening, While operations such as addition of liquids and reagents can be applied, liquids and substances can be circulated between the wells and the micro channels separated by the porous membrane.
  • the cell culture device is the cell culture device according to the first aspect or the second aspect, wherein the other of the respective microcavities is also a microchannel.
  • one of the pair of micro-cavities is a micro-channel and the other is a micro-channel, so that shear stress associated with the fluid flow should be applied to any surface of the porous membrane.
  • a cell culture device is the cell culture device according to any one of the first to fourth aspects, in which a plurality of porous membranes having the same opening diameter or different opening diameters are laminated. There is.
  • the strength can be increased by stacking a plurality of porous films.
  • the aperture ratio can be substantially reduced without changing the porosity, and the passage of cells through the porous membrane can be controlled.
  • a cell culture device is the cell culture device according to any one of the first to fifth aspects, wherein, as a membrane extension mechanism, a liquid flowing through the microchannel is used in the microchannel. It is equipped with an acceleration mechanism capable of increasing the flow velocity.
  • the membrane stretching mechanism stretches the porous membrane along the thickness direction
  • the pressure in the thickness direction generated by the liquid flowing in the micro flow channel can be applied to the porous membrane in the micro flow channel by the acceleration mechanism as such a mechanism.
  • the cell culture device is not limited to the cell culture device according to any one of the first to fifth aspects, in which at least the microchannel is formed in the pair of cavity members.
  • the membrane stretching mechanism includes a contact member that contacts the opposite side of the flexible cavity member to the side where the microchannel is formed, and the contact member along the thickness direction of the porous membrane. And a movable member capable of at least one of pressing and pulling.
  • the membrane stretching mechanism stretches the porous membrane along the thickness direction
  • the porous membrane can be stretched by changing the pressure of the microchannel by the mechanical configuration of the contact member and the movable member as such a mechanism.
  • At least one of the pair of cavity members in which the micro flow channel is formed has flexibility, and the membrane extension mechanism has the micro flow channel.
  • the membrane stretching mechanism stretches the porous membrane along the thickness direction
  • a mechanism that exerts a pressure (at least one of pressurization and depressurization) acting on the microchannel on the porous membrane for example, there is a mechanism that exerts a pressure (at least one of pressurization and depressurization) acting on the microchannel on the porous membrane.
  • the internal pressure change space presses the micro flow channel, and the porous membrane is pressed by this pressing.
  • the internal pressure change space pulls the microchannel, and the porous membrane is pulled along with this pulling. In either case, the porous membrane will stretch.
  • both pressurization and depressurization of these are possible.
  • a cell culture device is the cell culture device according to any one of the first to eighth aspects, which is provided on both sides of the microcavity and has flexibility in addition to the membrane extension mechanism.
  • a lateral extension mechanism having a cavity side wall and a lateral pressure change space provided outside each of the cavity side walls is provided, and the lateral extension mechanism has at least pressure and decompression in the lateral pressure change space.
  • FIG. 5 is a sectional view taken along line CC in FIG. 4. Another example of the porous membrane is shown in a sectional view taken along the line CC in FIG.
  • FIG. 2 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the cross section taken along line BB in FIG. 1.
  • FIG. 2 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the cross section taken along line BB in FIG. 1.
  • FIG. 2 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the cross section taken along the line BB in FIG. 1.
  • FIG. 2 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the cross section taken along the line BB in FIG. 1.
  • FIG. 2 is a sectional view taken along the line AA in FIG. 1.
  • FIG. 2 is a schematic diagram showing a state in which the porous film is not stretched in a part of the cross section taken along the line AA in FIG. 1.
  • FIG. 2 is a schematic view showing a state where the porous film is stretched in a part of the cross section taken along the line AA in FIG. 1.
  • FIG. 2 is a schematic view showing a state where the porous film is stretched in a part of the cross section taken along the line AA in FIG. 1.
  • It is a perspective view showing the whole cell culture device structure in a 2nd embodiment. It is an exploded perspective view showing the whole cell culture device structure in a 2nd embodiment.
  • FIG. 16 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the DD cross section in FIG. 15.
  • FIG. 16 is a schematic diagram showing an example of a state in which a cell layer is provided in a part of the DD cross section in FIG. 15.
  • FIG. 16 is a schematic view showing a state in which the porous film is not stretched in a part of the cross section taken along the line EE in FIG. 15.
  • FIG. 16 is a schematic view showing a state in which the porous film is stretched in a part of the cross section taken along the line EE in FIG. 15.
  • It is a perspective view showing the whole cell culture device structure in a 3rd embodiment. It is an exploded perspective view showing the whole cell culture device structure in a 3rd embodiment.
  • FIG. 23 is a schematic diagram showing a state in which the porous film is not expanded in a part of the cross section taken along the line FF in FIG. 22.
  • FIG. 23 is a schematic view showing a state where the porous film is stretched in a part of the cross section taken along the line FF in FIG. 22.
  • FIG. 23 is a schematic view showing a state where the porous film is stretched in a part of the cross section taken along the line FF in FIG. 22.
  • the cell culture device 10 of the present embodiment includes an upper cavity member 12 and a lower cavity member 14 facing each other as a pair of cavity members stacked in the thickness direction.
  • the cavity unit 16 is formed.
  • the upper cavity member 12 and the lower cavity member 14 are preferably made of a transparent transparent material such as PDMS (polydimethylsiloxane), for example.
  • the materials for the upper cavity member 12 and the lower cavity member 14 include PDMS (polydimethylsiloxane), epoxy resin, urethane resin, styrene thermoplastic elastomer, olefin thermoplastic elastomer, and acrylic resin. Examples thereof include thermoplastic elastomers and polyvinyl alcohol.
  • the rubber hardness of the upper cavity member 12 and the lower cavity member 14 is preferably 20 degrees or more and 80 degrees or less, and more preferably 50 degrees or more and 70 degrees or less.
  • the “rubber hardness” can be evaluated by measuring the hardness of the upper cavity member 12 and the lower cavity member 14 with a type A durometer according to the method specified in JIS K6253:2012. By having such rubber hardness, the upper cavity member 12 and the lower cavity member 14 have flexibility.
  • the concave portion 26 has an inflow port 26A, an outflow port 26B, and a flow path portion 26C that connects the inflow port 26A and the outflow port 26B.
  • the concave portion 20 has an inflow port 20A, an outflow port 20B, and a flow path section 20C that connects the inflow port 20A and the outflow port 20B.
  • the upper cavity member 12 is formed with through holes 22A and 22B which penetrate the upper cavity member 12 in the thickness direction and whose lower ends communicate with the inflow port 20A and the outflow port 20B, respectively.
  • the microcavity is a space having a size on the order of micrometers, which is defined by the facing surfaces of the pair of cavity members (the upper cavity member 12 and the lower cavity member 14).
  • the microcavity includes a microchannel (upper microchannel 18 and lower microchannel 24) that is a groove having a width of less than 1 mm, and a circular shape, an elliptical shape, a rectangular shape having a maximum diameter portion of less than 1 mm.
  • a well 19 (see FIGS. 20 and 21) described later, which is a hole having a polygonal shape and penetrating the cavity member.
  • the microcavities include an upper microcavity formed in the upper cavity member 12 and a lower microcavity formed in the lower cavity member 14.
  • both the upper microcavity and the lower microcavity have microchannels (the upper microchannel 18 and the lower microchannel). Road 24). Note that, in FIG. 3, configurations other than the upper cavity member 12, the lower cavity member 14, the upper microchannel 18, the lower microchannel 24, and the porous membrane 30 are omitted.
  • the inflow port 26A and the outflow port 26B of the lower cavity member 14 are provided at positions that do not overlap with the inflow port 20A and the outflow port 20B of the upper cavity member 12 in a plan view.
  • the flow passage portion 26C of the lower cavity member 14 is provided at a position overlapping the flow passage portion 20C of the upper cavity member 12 in a plan view.
  • the upper cavity member 12 is formed with through holes 28A and 28B which penetrate the upper cavity member 12 in the thickness direction and whose lower ends communicate with the inflow port 26A and the outflow port 26B of the lower cavity member 14, respectively. .. Further, on the outer peripheral surface of the cavity unit 16 (the upper cavity member 12 and the lower cavity member 14), recesses 29 are provided at positions where spacers 46 described later are arranged.
  • a porous film 30 is arranged between the facing surfaces 12A and 14A of the upper cavity member 12 and the lower cavity member 14.
  • the porous film 30 is made of, for example, a hydrophobic polymer that can be dissolved in a hydrophobic organic solvent.
  • the hydrophobic organic solvent is a liquid having a solubility in water at 25° C. of 10 (g/100 g water) or less.
  • hydrophobic polymer polystyrene, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyhexafluoropropene, polyvinyl ether, polyvinylcarbazole, polyvinyl acetate, polytetrachloride.
  • polyester eg polyethylene terephthalate, polyethylene naphthalate, polyethylene succinate, polybutylene succinate, polylactic acid, poly-3-hydroxybutyrate etc.
  • polylactone eg polycaprolactone etc.
  • polyamide or polyimide eg , Nylon, polyamic acid, etc.
  • polyurethane polyurea, polybutadiene, polycarbonate, polyaromatics, polysulfone, polyether sulfone, polysiloxane derivative, cellulose acylate (eg, triacetyl cellulose, cellulose acetate propionate, cellulose acetate butyrate). Rate) and other polymers.
  • any block copolymer that can be dissolved in a hydrophobic organic solvent can be used.
  • block copolymers that can be used in the present disclosure include styrene-butadiene block copolymers, styrene-isoprene block copolymers and other aromatic hydrocarbon-aliphatic hydrocarbon block copolymers, styrene-acrylic acid block copolymers, styrene-acrylic.
  • Aromatic hydrocarbon-aliphatic polar compound block copolymer such as sodium acid acid block copolymer, styrene-polyethylene glycol block copolymer, fluorene-methyl methacrylate block copolymer, aromatic hydrocarbon-aromatic polar compound block copolymer such as styrene-vinylpyridine Etc.
  • These polymers may be homopolymers, copolymers, polymer blends or polymer alloys, if necessary, from the viewpoints of solubility in solvents, optical properties, electrical properties, film strength, elasticity, etc. These polymers may be used alone or in combination of two or more.
  • the material of the porous film 30 is not limited to the hydrophobic polymer, and various materials can be selected from the viewpoint of cell adhesiveness and the like.
  • the upper surface 30A and the lower surface 30B of the porous film 30 cover the flow path portions 20C and 26C of the upper micro flow path 18 and the lower micro flow path 24, and separate the upper micro flow path 18 and the lower micro flow path 24 from each other. ..
  • the upper surface 30A of the porous membrane 30 defines the upper microchannel 18 together with the concave portion 20 of the upper cavity member 12, and the lower surface 30B of the porous membrane 30 forms the lower side together with the concave portion 26 of the lower cavity member 14.
  • a micro flow path 24 is defined.
  • the porous film 30 is formed with a plurality of in-membrane spaces 32 penetrating in the thickness direction, and the upper surface 30A and the lower surface 30B of the porous film 30 have inner spaces on both sides thereof.
  • 32 openings 32A are provided respectively.
  • the opening 32A is circular in plan view. The openings 32A are provided apart from each other, and the flat portion 34 extends between the openings 32A adjacent to each other.
  • the opening 32A is not limited to the circular shape, and may be a polygonal shape or an elliptical shape.
  • the plurality of openings 32A are arranged in a honeycomb shape as shown in FIG.
  • the honeycomb arrangement means that six openings 32A are equally arranged around an arbitrary opening 32A (excluding the openings 32A at the edge of the film), and the centers of the six openings 32A are regular hexagons. Is located at the apex of, and the center of the opening 32A located at the center corresponds to the center of the regular hexagon.
  • the term "equal distribution” does not necessarily mean that the central angle is 60° and that the six surrounding openings 32A are arranged at substantially equal intervals with respect to the central opening 32A. It should have been done.
  • the "center of the opening 32A" means the center of the opening 32A in plan view.
  • the inner space 32 of the porous film 30 has a sphere shape in which the upper and lower ends of the sphere are cut off. Note that the sphere referred to here does not need to be a true sphere, and distortion that is generally recognized as a sphere is acceptable.
  • the intra-membrane spaces 32 adjacent to each other have a lateral communication structure in which the communication holes 36 communicate with each other inside the porous film 30.
  • the lateral communication structure refers to a space structure in which adjacent membrane spaces 32 communicate with each other inside the porous membrane 30.
  • the term "horizontal” as used herein means a plane direction orthogonal to the vertical direction when the thickness direction of the porous film 30 is vertical. Since the openings 32A are arranged in a honeycomb shape in the porous film 30, the arbitrary intra-membrane space 32 communicates with all of the six intra-membrane spaces 32 that are evenly arranged around it.
  • the intra-membrane space 32 may have a barrel shape, a columnar shape, a polygonal prism shape, or the like, and the communication hole 36 may have a cylindrical void that connects adjacent intra-membrane spaces 32. ..
  • the opening 32A of the porous film 30 may be provided on one surface, for example, only the upper surface 30A of the porous film 30 (or only the lower surface 30B).
  • Such a porous membrane 30 having the openings 32A provided on only one surface is suitable for culturing cells in the intramembrane space 32.
  • the region of the upper surface 30A and the lower surface 30B of the porous membrane 30 on which cells are seeded is fibronectin, collagen (eg, type I collagen, IV Type collagen, or type V collagen), laminin, vitronectin, gelatin, perlecan, nidogen, proteoglycan, osteopontin, tenascin, nephronectin, basement membrane matrix and polylysine, preferably at least one selected from the group consisting of ..
  • collagen eg, type I collagen, IV Type collagen, or type V collagen
  • the cell culture device 10 of the present embodiment is used as an organ simulating device or the like, by providing a cell layer seeded with cells constituting the organ to be simulated on at least one of the upper surface 30A and the lower surface 30B of the porous membrane 30.
  • the inside of the upper micro-channel 18 and the inside of the lower micro-channel 24 can be made to have an environment close to that of the organ to be simulated.
  • the cell layer 100 can be provided on the upper surface 30A of the porous membrane 30. Further, as shown in FIG. 8, the cell layer 100 may be provided on the lower surface 30B of the porous film 30.
  • the cell layer 100 can be provided on both the upper surface 30A and the lower surface 30B of the porous film 30.
  • the cell layers 100 provided on the upper surface 30A and the lower surface 30B may be formed by seeding cells of the same type, or may be formed by seeding cells of different types.
  • the diameter of the openings 32A be 2 to 5 ⁇ m, which is smaller than the diameter of the cells.
  • the film thickness is preferably 2 to 20 ⁇ m.
  • the cell layer 100 can be provided in the inner space 32 of the porous membrane 30.
  • the porous film 30 in which the intra-membrane space 32 is open only on one surface is desirable.
  • the film thickness is preferably 20 to 200 ⁇ m.
  • which of the upper surface 30A, the lower surface 30B of the porous film 30 and the intramembrane space 32 is provided with the cell layer 100 can be appropriately selected according to the characteristics of the cells constituting the organ to be simulated. ..
  • the dew condensation method is a method in which the surface of the material forming the porous film 30 is condensed to form the film inner space 32 by using water drops as a template.
  • the dew condensation method can reduce the film thickness of the porous film 30 and increase the porosity and the opening rate of the openings 32A as compared with other methods. Specifically, a film thickness of 0.5 to 10 ⁇ m is possible by the condensation method. Further, the communication hole 36 can be provided in the porous film 30. Therefore, in the present embodiment, the porous film 30 is manufactured by the dew condensation method.
  • the opening ratio can be defined as the ratio of the area of the openings 32A to the area of the porous film 30.
  • a means for forming the present porous membrane other than the dew condensation method there is an emulsification method using a fine oil droplet or a fine water droplet created by emulsification as a template, instead of a water droplet derived from dew condensation, and details thereof are disclosed in International Publication 2017/104610. No. 6,096,242, the contents of which are included in the present disclosure.
  • the average opening diameter of the openings 32A is 1 ⁇ m or more and 200 ⁇ m or less.
  • the average opening diameter of the openings 32A is 1 ⁇ m or more, it is easy to form the lateral communication structure of the intramembrane space 32.
  • the average opening diameter of the openings 32A is 200 ⁇ m or less, it is easy to maintain the honeycomb-shaped arrangement without the adjacent openings 32A fusing. Therefore, a suitable average opening diameter of the openings 32A is 1 ⁇ m or more and 200 ⁇ m or less.
  • the average opening diameter means the average value of the diameters of the plurality of openings 32A on the surface of the porous film 30. This average opening diameter can be, for example, the average value of the values obtained by observing the surface of the porous film 30 under a microscope and measuring the diameters of a considerable number of the openings 32A.
  • the porosity of the porous film 30 is 40% or more and 90% or less.
  • the porosity of the porous film 30 is 40% or more, it is easy to form the lateral communication structure of the intra-membrane space 32.
  • the porosity of the porous film 30 is 90% or less, it is easy to maintain the shape of the porous film 30, and the strength is not lowered and the porous film 30 is not easily broken. Therefore, the preferable porosity of the porous film 30 is 40% or more and 90% or less.
  • the porosity refers to the ratio of the volume of the intra-membrane space 32 to the volume of the porous membrane 30.
  • the porosity is obtained by, for example, observing a cross section of the porous film 30 under a microscope and estimating the observed intra-membrane space 32 as a spherical trapezoidal shape in which two upper and lower sides and six side faces are cut off in a circular shape.
  • the volume of the plurality of in-membrane spaces 32 can be calculated as a percentage obtained by dividing the volume of the porous membrane 30 in which the in-membrane spaces 32 exist.
  • the film thickness of the porous film 30 is 0.5 ⁇ m or more and 100 ⁇ m or less.
  • the numerical value of the film thickness is such that the aspect ratio between the opening diameter of the opening 32A and the height of the intra-membrane space 32 (that is, the value obtained by dividing the opening diameter of the opening 32A by the height of the intra-membrane space 32). It is a numerical value derived from the fact that exceeding 2 is practically impossible.
  • the film thickness is preferably 0.5 to 10 ⁇ m.
  • the total film thickness of the porous films 30 is preferably 10 to 200 ⁇ m.
  • the tensile force per unit width required for 20% elongation of the porous film 30 is 0.1 N/m or more and 20 N/m or less because the porous film 30 has the numerical range as described above. it can.
  • the numerical range of the tensile force is a value that can be realized by the difference in pressure generated by the liquid flowing through the upper microchannel 18 and the lower microchannel 24.
  • the cell culture device 10 has a pair of holding plates 38 as holding members for holding the cavity unit 16 in a compressed state in the thickness direction.
  • the pair of holding plates 38 is provided separately from the cavity unit 16 at both ends in the thickness direction of the cavity unit 16, that is, above the upper cavity member 12 and below the lower cavity member 14. Is sized to cover the entire upper surface and the entire lower surface of the lower cavity member 14.
  • a plurality (eight in this embodiment) of bolt holes 40 that penetrate in the thickness direction are formed at positions corresponding to each other in the pair of holding plates 38.
  • the holding plate 38 provided on the upper side of the upper cavity member 12 is formed with through holes 42A, 42B, 44A, 44B which communicate with the through holes 22A, 22B, 28A, 28B of the upper cavity member 12, respectively. ing.
  • inflow tubes 62A and 64A are connected to the through holes 42A and 44A, respectively, and outflow tubes 62B and 64B are connected to the through holes 42B and 44B, respectively.
  • a solution, a cell suspension or the like flows into the upper microchannel 18 and the lower microchannel 24 through the inflow tubes 62A and 64A, and the solution flows from the upper microchannel 18 and the lower microchannel 24 through the outflow tubes 62B and 64B. And cell suspension etc. flow out.
  • An accelerating mechanism 70 as a membrane stretching mechanism is connected upstream of the inflow tube 64A flowing into the lower microchannel 24.
  • an accelerating mechanism 75 which is also a membrane stretching mechanism, is connected upstream of the inflow tube 62A that flows into the upper microchannel 18. That is, the acceleration mechanisms 70 and 75 as the film stretching mechanism are connected to the lower cavity member 14 and the upper cavity member 12 via the inflow tubes 64A and 62A, respectively.
  • These accelerating mechanisms 70 and 75 are, for example, a circulation pump capable of changing the flow velocity of the liquid flowing through the lower microchannel 24 and the upper microchannel 18, and the lower microchannel 24 and the upper microchannel 18. It can be configured by a pressure adjuster or the like that pressurizes the liquid filled in from the upstream side.
  • gas or liquid is injected from the upstream side of at least one of the corresponding inflow tubes 62A and 64A in a state where at least one of the outflow tubes 62B and 64B is closed.
  • a pressure may be applied.
  • a pressure resistance adjusting mechanism is connected to the downstream side of at least one of the outflow tubes 62B and 64B, and control is performed so that the cross-sectional area of at least one of the outflow tubes 62B and 64B is expanded or reduced, thereby achieving the same transmission. It is also possible to adopt a configuration in which the pressure inside the microchannel can be adjusted even with the liquid amount.
  • a plurality of (eight in this embodiment) spacers 46 that define the spacing between the holding plates 38 are provided outside the recess 29 of the cavity unit 16 between the pair of holding plates 38.
  • the spacers 46 are cylindrical members each having an inner diameter substantially the same as the inner diameter of the bolt hole 40, and are arranged at positions corresponding to the bolt hole 40.
  • the pair of holding plates 38 are joined to each other by a plurality of bolts 50 that are inserted into the bolt holes 40 and the spacers 46 and fixed with the nuts 48. At this time, the upper cavity member 12 and the lower cavity member 14 are compressed and held by the pair of holding plates 38 with the porous film 30 sandwiched therebetween.
  • FIGS. 12 to 14 show cross-sections taken along the line AA of FIG. 1 except for the upper cavity member 12, the lower cavity member 14, the upper microchannel 18, the lower microchannel 24, and the porous membrane 30. It is omitted.
  • the liquid flowing through the upper microchannel 18 and the lower microchannel 24 applies shear stress in the direction of the horizontal arrow as shown in FIG.
  • pressure in the thickness direction is generated by the liquid flowing through the upper microchannel 18 and the lower microchannel 24. This pressure is determined by the viscosity of the liquid, the flow velocity and the shape of the flow channel.
  • the upper microchannel 18 and the lower microchannel 24 have the same pressure and no pressure difference occurs, so that the porous membrane 30 does not expand.
  • the pressure in the thickness direction also changes due to the change in the flow velocity. Change.
  • the pressure in the lower microchannel 24 becomes larger than the pressure in the upper microchannel 18, a pressure difference is generated between the upper and lower microchannels.
  • pressure in the thickness direction is applied to the porous membrane 30 from the lower microchannel 24, and the porous membrane 30 expands upward along the thickness direction. Also, the shear stress in the direction of the horizontal arrow continues to be applied.
  • the flow velocity of the liquid flowing in the upper micro flow channel 18 is controlled by the acceleration mechanism 75 to be higher than the flow velocity of the liquid flowing in the lower micro flow channel 24, so that the pressure in the upper micro flow channel 18 becomes lower.
  • the pressure in the thickness direction from the upper micro flow channel 18 is applied to the porous film 30, and the porous film 30 moves downward in the thickness direction. Extend to. Also, the shear stress in the direction of the horizontal arrow continues to be applied.
  • the thickness direction in the thickness direction caused by the pressure difference due to the liquid flowing through the upper microchannel 18 and the lower microchannel 24 is increased.
  • the porous film 30 can be expanded in the thickness direction toward the side to which pressure is applied.
  • the porous membrane 30 has a cell layer 100 formed at an appropriate position as illustrated in FIGS. 7 to 10.
  • the pressure in the thickness direction applied to the porous membrane 30 as described above causes the porous membrane 30 to expand, and a vertical force is applied to the cell layer 100. Further, due to the shear stress of the fluid, a horizontal force is also applied to the cell layer 100.
  • the cell layer 100 is given a mechanical stress simulating the inside of a living body, and it becomes possible to promote orientation control and maturation of cells. This pressure in the thickness direction can occur even at a low flow velocity, and the porous membrane 30 in the cell culture device 10 of the present disclosure can be expanded by 20% even with such a pressure in the thickness direction. ..
  • intestinal epithelial cells are seeded on the porous membrane 30 as cells
  • cardiomyocytes are seeded on the porous membrane 30 as cells, mechanical stress on the porous membrane 30 can promote autonomous pulsation of the cardiomyocytes.
  • the cell culture device 10 of the present embodiment includes an upper cavity member 12 and a lower cavity member 14 facing each other as a pair of cavity members stacked in the thickness direction.
  • the cavity unit 16 is formed.
  • the material and physical properties of the upper cavity member 12 and the lower cavity member 14 are the same as in the first embodiment.
  • a recess 26 that defines the lower microchannel 24 as one of the microcavities is formed on the upper surface of the lower cavity member 14, that is, the surface 14A facing the upper cavity member 12.
  • the concave portion 26 has an inflow port 26A, an outflow port 26B, and a flow path portion 26C that connects the inflow port 26A and the outflow port 26B.
  • the upper cavity member 12 is formed with two wells 19 as the other of the microcavities so as to penetrate from the upper surface to the surface 12A facing the lower cavity member 14.
  • the number of wells 19 is not limited to two, and may be one or three or more.
  • the well 19 is a closed space having no inflow port and outflow port other than the upper opening 19A and the lower opening 19B.
  • the well 19 is formed as the upper microcavity in the upper cavity member 12, and the lower microchannel 24 is formed in the lower cavity member 14 as the lower microcavity.
  • the well 19 and the micro flow channel are as described in the first embodiment.
  • the upper microcavity is the well 19 and the lower microcavity is the lower microchannel 24, as shown in FIG. 17 that schematically shows a cross section taken along line DD of FIG. All the wells 19 of the upper cavity member 12 are provided at positions where they overlap the flow passage portion 26C of the lower cavity member 14 in a plan view.
  • the components other than the upper cavity member 12, the lower cavity member 14, the upper microchannel 18, the lower microchannel 24, and the porous membrane 30 are omitted.
  • the porous membrane 30 arranged between the upper cavity member 12 and the lower cavity member 14 is the same as in the first embodiment.
  • the lower end of the well 19 is defined by the porous film 30.
  • the upper end of the well 19 is open upward. From the upper end of the well 19, cells can be seeded and solutions and reagents can be added.
  • the cell layer 100 is formed on the upper surface 30A of the porous membrane 30, and the solution 200 is formed up to the middle portion of the lower microchannel 24 and the well 19.
  • the solution and the reagent can be appropriately added from the upper end of the well 19 while culturing the cell layer 100 in the well 19.
  • the electrode 300 by attaching the electrode 300 to the cell layer 100, it is possible to apply an electrical stimulation to the cell layer 100 and measure the potential generated in the cell layer 100.
  • the cardiomyocytes can be seeded on the porous membrane 30 to simulate the electrical dynamics of the cardiomyocytes.
  • various sensors for example, a temperature sensor, a pressure sensor, a chemical sensor, etc. may be attached instead of the electrodes to measure the temperature and pressure or various chemical substances.
  • the holding member is almost the same as in the first embodiment. However, as shown in FIG. 15 in a state where the cavity unit 16 is held by the holding member, the inflow tube 64A is connected to the through hole 44A, and the outflow tube 64B is connected to the through hole 44B. A solution, a cell suspension or the like flows into the lower micro flow channel 24 through the inflow tube 64A, and a solution, a cell suspension or the like flows out from the lower micro flow channel 24 through the outflow tube 64B.
  • two through holes 45 corresponding to the opening 19A are provided substantially in the center of the upper holding plate 38.
  • An accelerating mechanism 70 as a membrane stretching mechanism is connected upstream of the inflow tube 64A flowing into the lower microchannel 24. That is, the acceleration mechanism 70 as the film stretching mechanism is connected to the lower cavity member 14 via the inflow tube 64A.
  • the acceleration mechanism 70 is the same as in the first embodiment.
  • FIG. 20 shows a state in which the flow velocity of the liquid flowing through the lower microchannel 24 is so small that the pressure in the thickness direction is not applied to the extent that the porous film 30 extends.
  • the flow velocity of the liquid flowing through the lower microchannel 24 is increased by the acceleration mechanism 70, the pressure in the thickness direction from the lower microchannel 24 is applied to the porous membrane 30 as shown in FIG. Is added, the porous film 30 extends downward along the thickness direction.
  • the flow velocity of the liquid referred to here is determined by the elongation property and area of the porous membrane, the viscosity of the liquid, and the like.
  • the pressure in the thickness direction generated by the change in the flow velocity of the liquid flowing through the lower microchannel 24 is applied.
  • the porous film 30 can be expanded along the thickness direction.
  • the cell culture device 10 of the present embodiment is composed of an upper cavity member 12 and a lower cavity member 14 facing each other as a pair of cavity members stacked in the thickness direction.
  • the cavity unit 16 is formed.
  • the materials, physical properties, and structures of the upper cavity member 12 and the lower cavity member 14 are the same as in the first embodiment.
  • porous membrane 30 arranged between the upper cavity member 12 and the lower cavity member 14 is also the same as in the first embodiment.
  • the holding member is almost the same as that of the first embodiment, but is different in that a rectangular contact opening 39 is formed substantially at the center of the lower holding plate 38 of the pair of holding plates 38.
  • a contact member 80 having the same shape is fitted in the contact opening 39.
  • the upper surface of the fitted contact member 80 is the lower surface of the lower cavity member 14 which is the opposite side of the flexible lower cavity member 14 to the side where the flow path portion 26C of the lower micro flow path 24 is formed. It is fixed in contact.
  • a movable member 85 that can move up and down by a power source (not shown) is fixed to the lower surface of the contact member 80. As the movable member 85 moves up and down, the contact member 80 also moves up and down, and the lower cavity member 14 is also deformed accordingly. That is, the contact member 80 and the movable member 85 function as a film stretching mechanism. In other words, the contact member 80 as the film stretching mechanism is connected to the lower cavity member 14.
  • inflow tubes 62A and 64A are connected to the through holes 42A and 44A, respectively, and outflow tubes 62B and 64B are connected to the through holes 42B and 44B, respectively.
  • a solution, a cell suspension or the like flows into the upper microchannel 18 and the lower microchannel 24 through the inflow tubes 62A and 64A, and the solution flows from the upper microchannel 18 and the lower microchannel 24 through the outflow tubes 62B and 64B. And cell suspension etc. flow out.
  • the contact member 80 and the movable member 85 are provided as the membrane stretching mechanism, and the membrane stretching mechanism causes the porous membrane 30 to stretch.
  • 24 to 26 are sectional views taken along the line FF of FIG. 22, showing the upper cavity member 12 and the lower cavity member 14, the upper microchannel 18 and the lower microchannel 24, the porous membrane 30, the contact member 80 and the movable member.
  • the configurations other than the member 85 are omitted.
  • the contact member 80 when the movable member 85 operates and moves upward along the thickness direction of the porous membrane 30, the contact member 80 also moves upward at the same time. Along with this, the contact member 80 presses the lower cavity member 14 upward, and thus the lower cavity member 14 undergoes upward deformation. This deformation is applied as a pressing force to the porous membrane 30 via the solution in the lower microchannel 24, so that the porous membrane 30 extends upward.
  • the contact member 80 when the movable member 85 operates and moves downward along the thickness direction of the porous film 30, the contact member 80 also moves downward. Along with that, the contact member 80 pulls the lower cavity member 14 downward, so that the lower cavity member 14 undergoes downward deformation. This deformation is applied as a traction force to the porous membrane 30 via the solution in the lower microchannel 24, so that the porous membrane 30 extends downward.
  • the contact member 80 and the movable member 85 as the film stretching mechanism can be provided in the upper cavity member 12 as in the modification of the present embodiment shown in FIGS. 27 to 29. That is, of the pair of holding plates 38, a contact opening (not shown) similar to the contact opening 39 shown in FIG. 23 is provided substantially at the center of the upper holding plate 38, and the contact member having the same shape is provided in this contact opening. Insert 80.
  • the lower surface of the fitted contact member 80 is fixed in contact with the upper surface of the upper cavity member 12, which is the side of the flexible upper cavity member 12 opposite to the side on which the upper microchannel 18 is formed. That is, the contact member 80 as the film stretching mechanism is connected to the upper cavity member 12.
  • a movable member 85 that can move up and down by a power source (not shown) is fixed to the upper surface of the contact member 80. As the movable member 85 moves up and down, the contact member 80 also moves up and down, and the upper cavity member 12 is also deformed accordingly.
  • 27 to 29 show cross sections from the same viewpoint as FIGS. 24 to 26.
  • the contact member 80 when the movable member 85 operates and moves downward along the thickness direction of the porous membrane 30, the contact member 80 also moves downward at the same time. Along with that, the contact member 80 presses the upper cavity member 12 downward, so that the upper cavity member 12 undergoes downward deformation. This deformation is applied as a pressing force to the porous membrane 30 via the solution in the upper microchannel 18, whereby the porous membrane 30 extends downward along the thickness direction.
  • the contact member 80 when the movable member 85 operates and moves upward along the thickness direction of the porous film 30, the contact member 80 also moves upward at the same time. Along with that, the contact member 80 pulls the upper cavity member 12 upward, so that the upper cavity member 12 undergoes downward deformation. This deformation is applied as a traction force to the porous membrane 30 via the solution in the upper microchannel 18, whereby the porous membrane 30 extends upward along the thickness direction.
  • a part of the contact member 80 as a membrane extension mechanism can be fitted into the lower cavity member 14 and directly connected to the porous membrane 30.
  • the “flexibility” in the present embodiment means that the material forming the lower cavity member 14 or the upper cavity member 12 has such a softness that it can be deformed by being pressed by the contact member 80. means.
  • [Fourth Embodiment] 31 to 33 show a part of the cell culture device 10 according to the fourth embodiment in a sectional view according to the section taken along the line BB of FIG. 1, and show the upper cavity member 12 and the lower cavity member. Structures other than the cavity unit 16 composed of 14 and the porous film 30 are omitted. The upper microchannel 18 formed in the upper cavity member 12 and the lower microchannel 24 formed in the lower cavity member 14 are separated by a porous film 30.
  • an internal pressure change space 90 as a membrane extension mechanism is formed inside the lower cavity member 14.
  • the internal pressure change space 90 is formed along the lower microchannel 24 and is connected to an external pressurizing/depressurizing mechanism (pressurizing pump and vacuum pump) not shown.
  • the internal pressure change space 90 When a gas is sent to the internal pressure change space 90 from a pressurizing pump as a pressurizing/depressurizing mechanism to pressurize it, the internal pressure change space 90 expands toward the lower micro flow channel 24, as shown in FIG. 32. This expansion presses the side of the lower cavity member 14 opposite to the side where the lower microchannel 24 is formed, and acts as a pressing force on the porous membrane 30 via the solution in the lower microchannel 24. By adding, the porous film 30 expands upward along the thickness direction.
  • the internal pressure change space 90 contracts as shown in FIG. Due to this contraction, the side of the lower cavity member 14 opposite to the side where the lower microchannel 24 is formed is pulled, and is applied as a pulling force to the porous membrane 30 via the solution in the lower microchannel 24. Then, the porous film 30 extends downward along the thickness direction.
  • the internal pressure change space 90 may be formed inside the upper cavity member 12.
  • the expansion of the internal pressure change space 90 presses the side of the upper cavity member 12 opposite to the side on which the upper microchannel 18 is formed, and permeates through the solution in the upper microchannel 18.
  • the porous membrane 30 extends downward along the thickness direction.
  • the contraction of the internal pressure change space 90 pulls the side of the upper cavity member 12 opposite to the side on which the upper microchannel 18 is formed, and the porous membrane is mediated by the solution in the upper microchannel 18.
  • the porous film 30 extends upward along the thickness direction.
  • the “flexibility” in the present embodiment means that the material forming the lower cavity member 14 or the upper cavity member 12 has a hardness that allows the material to be deformed by the expansion or contraction of the internal pressure change space 90.
  • [Fifth Embodiment] 34 to 36 show a part of the cell culture device 10 according to the fifth embodiment in a sectional view according to the section taken along the line BB of FIG. 1, and show the upper cavity member 12 and the lower cavity member. Structures other than the cavity unit 16 composed of 14 and the porous film 30 are omitted. The upper microchannel 18 formed in the upper cavity member 12 and the lower microchannel 24 formed in the lower cavity member 14 are separated by a porous film 30.
  • flexible cavity sidewalls 95A are provided on both sides of the upper microchannel 18 and the lower microchannel 24 as the microcavities.
  • the term “both sides of the microcavity” means the sides parallel to both the flow channel direction of the microchannel and the facing direction of the microcavity.
  • a lateral pressure change space 95B as a space extending between the upper cavity member 12 and the lower cavity member 14 is provided outside each of the cavity side walls 95A.
  • the cavity side wall 95A and the lateral pressure change space 95B form a lateral extension mechanism 95.
  • the porous film 30 is connected to the cavity side wall 95A as the lateral extension mechanism 95.
  • the lateral pressure change space 95B is formed along the upper microchannel 18 and the lower microchannel 24, and is connected to an external pressurizing/depressurizing mechanism (pressurizing pump and vacuum pump) not shown. .
  • the "width direction of the porous membrane” means a direction perpendicular to both the flow channel direction of the micro flow channel and the opposing direction of the microcavity.
  • the term “flexibility” in the present embodiment means that the material forming the upper cavity member 12 and the lower cavity member 14 has such a hardness that it can be deformed by expansion or contraction of the lateral pressure change space 95B.
  • the porous film 30 has been used as a single layer, but a plurality of porous films 30 may be laminated and used.
  • a plurality of porous films 30 may be laminated and used.
  • FIG. 37 when two porous films 30 having the same diameter opening 32A and the same thickness are laminated and used with the positions of the openings 32A aligned, the stretching property is improved. The strength can be increased while keeping it.
  • substantially lowering the aperture ratio means that when the laminated porous film 30 is projected in a plan view, the openings 32A of a certain layer are covered with a substantial portion between all the openings 32A adjacent to each other. It means that the area occupied by the opening 32A is reduced.
  • porous membranes 30 having openings 32A having the same diameter and having the same thickness can be laminated.
  • FIG. 40 it is also possible to stack and use a plurality of types of porous membranes having different diameters and thicknesses of the openings 32A.
  • the number and type of the porous membranes 30 to be laminated and how to shift the positions of the openings 32A can be appropriately determined depending on the usage mode, for example, the size of the cell culture device 10, the cell type used, the type of experiment, and the like.
  • FIG. 41 In the example of the porous film used in the present disclosure, as shown in the plan view of FIG. 41, circular openings are regularly arranged in a honeycomb shape.
  • This porous film is made of polybutadiene (PB, Example 1) or polycarbonate (PC, Example 2), and its cross section is as shown in FIG. 5, and the upper and lower openings have a lateral communication structure. It penetrates vertically by the inner space.
  • the thickness of this porous film was 3 ⁇ m, the opening diameter was 5 ⁇ m, the porosity was 85%, and the opening ratio was 63%.
  • three layers of PB-made porous membranes were used as Example 3
  • three layers of PC-made porous membranes were used as Example 4.
  • the porous film used as a comparative example is made of PC and has circular openings sparsely and irregularly as shown in the plan view photograph of FIG. 43.
  • the cross section of this porous film is as shown in FIG. 44, and the directions and lengths of the holes penetrate are different.
  • the thickness of this porous film is 10 ⁇ m, the opening diameter is 5 ⁇ m, the porosity is 10%, and the opening ratio is 3 to 10%.
  • the porous membrane of Example 2 made of the same material as that of Comparative Example had a tensile force of 2.48 N/m per unit width required for 20% elongation, which was a comparative example (472.5 N/m). It was possible to stretch with a force as small as about 0.5% of m).
  • the porous film of Example 2 was laminated in three layers, the film thickness was almost the same as that of the comparative example, but the tensile force per unit width required for 20% elongation was 7.5 N/m. It was possible to stretch with a small force of about 1.6%.
  • FIG. 46 is an enlarged view of the vicinity of the X axis in FIG. 45.
  • the porous membrane of the comparative example reached the yield point in the vicinity of the tensile force per unit width exceeding 400 N/m, and ruptured when the tensile force per unit width showed an elongation of about 65% near 550 N/m ( Figure 45).
  • the yield point was reached near a tensile force of 1.5 N/m per unit width, and about 88% was reached near a tensile force of 4.3 N/m per unit width. It broke when it showed elongation (Fig. 46).
  • the porous membrane of Example 1 was substantially elastically deformed until it showed an elongation of about 180% at a tensile force of about 0.5 N/m per unit width, and was fractured soon (FIG. 46). As described above, it was shown that the porous membranes of the examples can be deformed with a smaller tensile force than the comparative examples.

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Abstract

Ce dispositif de culture cellulaire est pourvu d'une paire d'éléments de cavité se faisant face et possédant chacun une microcavité formée sur les surfaces en vis-à-vis, d'un film poreux séparant les microcavités et disposant d'ouvertures agencées en un motif en nid d'abeilles sur une ou les deux surfaces, et d'un mécanisme d'étirement de film étant relié à l'élément de cavité ou au film poreux, et qui peut étirer le film poreux au moins dans la direction de l'épaisseur par application d'une force sur le film poreux dans au moins la direction de l'épaisseur. Le film poreux comporte des espaces intramembranaires communiquant à partir des ouvertures dans la direction de l'épaisseur, et possède une structure liée horizontalement dans laquelle les espaces intramembranaires adjacents communiquent l'un avec l'autre à l'intérieur du film poreux. Le diamètre d'ouverture moyen des ouvertures est de 1 à 200 µm, le taux de porosité est de 40 à 90 %, l'épaisseur de la membrane est de 0,5 à 100 µm, et la force de traction par unité de largeur nécessaire pour l'étirement de 20 % est de 0,1 à 20 N/m.
PCT/JP2019/046230 2018-12-06 2019-11-26 Dispositif de culture cellulaire WO2020116254A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462564A (zh) * 2021-06-25 2021-10-01 上海睿钰生物科技有限公司 培养装置

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JPS5415538B2 (fr) * 1974-03-25 1979-06-15
WO2009099066A1 (fr) * 2008-02-04 2009-08-13 Shimadzu Corporation Biodispositif
WO2009139177A1 (fr) * 2008-05-15 2009-11-19 国立大学法人大阪大学 Procédé d'induction de plaquettes sanguines
JP2014506801A (ja) * 2011-02-28 2014-03-20 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 細胞培養システム
JP2015516154A (ja) * 2012-04-18 2015-06-11 ヘモシアー・リミテッド・ライアビリティ・カンパニーＨｅｍｏＳｈｅａｒ， ＬＬＣ 病理学的状態または生理学的状態に対するＩｎｖｉｔｒｏモデル
JP2016535591A (ja) * 2013-10-21 2016-11-17 ヘモシアー・リミテッド・ライアビリティ・カンパニーＨｅｍｏＳｈｅａｒ， ＬＬＣ 腫瘍微細環境のための試験管内モデル
WO2018061846A1 (fr) * 2016-09-27 2018-04-05 富士フイルム株式会社 Procédé de production de tissu cellulaire, et film poreux
WO2018096054A1 (fr) * 2016-11-24 2018-05-31 Alveolix Ag Système et procédé de culture cellulaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5415538B2 (fr) * 1974-03-25 1979-06-15
WO2009099066A1 (fr) * 2008-02-04 2009-08-13 Shimadzu Corporation Biodispositif
WO2009139177A1 (fr) * 2008-05-15 2009-11-19 国立大学法人大阪大学 Procédé d'induction de plaquettes sanguines
JP2014506801A (ja) * 2011-02-28 2014-03-20 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 細胞培養システム
JP2015516154A (ja) * 2012-04-18 2015-06-11 ヘモシアー・リミテッド・ライアビリティ・カンパニーＨｅｍｏＳｈｅａｒ， ＬＬＣ 病理学的状態または生理学的状態に対するＩｎｖｉｔｒｏモデル
JP2016535591A (ja) * 2013-10-21 2016-11-17 ヘモシアー・リミテッド・ライアビリティ・カンパニーＨｅｍｏＳｈｅａｒ， ＬＬＣ 腫瘍微細環境のための試験管内モデル
WO2018061846A1 (fr) * 2016-09-27 2018-04-05 富士フイルム株式会社 Procédé de production de tissu cellulaire, et film poreux
WO2018096054A1 (fr) * 2016-11-24 2018-05-31 Alveolix Ag Système et procédé de culture cellulaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462564A (zh) * 2021-06-25 2021-10-01 上海睿钰生物科技有限公司 培养装置
CN113462564B (zh) * 2021-06-25 2022-04-01 上海睿钰生物科技有限公司 培养装置

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