WO2020116254A1

WO2020116254A1 - Cell culture device

Info

Publication number: WO2020116254A1
Application number: PCT/JP2019/046230
Authority: WO
Inventors: 晃寿伊藤; 圭介奥; 孝浩大場
Original assignee: 富士フイルム株式会社
Priority date: 2018-12-06
Filing date: 2019-11-26
Publication date: 2020-06-11

Abstract

This cell culture device is provided with a pair of cavity members which face each other and which each has a microcavity formed on the facing surfaces, a porous film which separates the microcavities and which has openings arranged in a honeycomb pattern on one or both surfaces, and a film stretching mechanism which is connected to the cavity member or the porous film, and which can stretch the porous film at least in the thickness direction by applying a force to the porous film in at least the thickness direction. The porous film has in-membrane spaces that communicate from the openings in the thickness direction, and has a horizontally linked structure in which the adjacent in-membrane spaces communicate with each other inside of the porous film. The average opening diameter of the openings is 1-200 μm, the void ratio is 40-90%, the membrane thickness 0.5-100 μm, and the tensile force per unit width necessary for 20% stretching is 0.1-20 N/m.

Description

Cell culture device

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).

In addition, as in Japanese Patent No. 5415538, Japanese Patent No. 5815643, and Japanese Unexamined Patent Application Publication No. 2017-136082, 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.

In recent years, 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. In 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. Further, as disclosed in Japanese Patent Nos. 5415538, 5815643, and Japanese Unexamined Patent Application Publication No. 2017-136082, 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.

However, in cell culture devices using porous membranes up to now, 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 according to a first aspect of the present disclosure 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. And 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. It has a lateral communication structure in which it communicates with each other inside the membrane, the average opening diameter of the openings is 1 μm or more and 200 μm or less, the porosity of the porous membrane is 40% or more and 90% or less, and the thickness of the porous membrane is 0.5 μm or more and 100 μm or less, the tensile force per unit width required for 20% extension of the porous membrane is 0.1 N/m or more and 20 N/m or less, and at least one of the microcavities has a microchannel. Is. In addition, extending a porous film at least along a thickness direction means extending up or down along a thickness direction.

According to the above configuration, 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 μm or more and 200 μm 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 μm or more and 100 μm 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. By stretching such a porous membrane at least along the thickness direction by a membrane stretching mechanism, cells that are cultivated on the surface of the porous membrane or in the inner space of the membrane are subjected to mechanical stress that is applied in vivo. Can be given.

A cell culture device according to a second aspect of the present disclosure 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.

According to the above configuration, since 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 according to the third aspect of the present disclosure 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.

According to the above configuration, 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 according to the fourth aspect of the present disclosure 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.

According to the above configuration, 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. You can

A cell culture device according to a fifth aspect of the present disclosure 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.

According to the above configuration, the strength can be increased by stacking a plurality of porous films. In addition, by arranging a plurality of porous membranes by shifting the positions of the openings, 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 according to a sixth aspect of the present disclosure 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.

As an aspect in which the membrane stretching mechanism stretches the porous membrane along the thickness direction, for example, there is a mechanism that uses the pressure in the thickness direction exerted by the liquid flowing through the microchannel on the porous membrane. According to the above configuration, 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 according to the seventh aspect of the present disclosure 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. In addition to having flexibility, 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.

As an aspect in which the membrane stretching mechanism stretches the porous membrane along the thickness direction, for example, there is a mechanism that uses pressure (at least one of pressurization and depressurization) that acts on the microchannel. According to the above configuration, 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.

In the cell culture device according to the eighth aspect of the present disclosure, 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. An internal pressure change space formed inside the cavity member, in which at least one of pressing by pressure and pulling by depressurization is possible along the thickness direction of the porous membrane from the side opposite to the side where the microchannel is formed. Equipped with.

As an aspect in which the membrane stretching mechanism stretches the porous membrane along the thickness direction, 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. According to the above configuration, by pressurizing the internal pressure change space as such a mechanism, the internal pressure change space presses the micro flow channel, and the porous membrane is pressed by this pressing. Alternatively, by depressurizing the internal pressure change space, 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. Alternatively, both pressurization and depressurization of these are possible.

A cell culture device according to a ninth aspect of the present disclosure 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. By performing one of these, at least one of pressing by pressure and pulling by depressurization is performed on the side wall of the cavity, thereby extending the porous film in the width direction.

According to the above configuration, in addition to the expansion in the thickness direction of the porous film, the expansion in the width direction of the porous film is possible.

According to the present disclosure, by using a porous membrane that can be stretched with a force smaller than the force required for stretching the porous membrane that has been used in the prior art, mechanical stress imparted to cells in vivo is It is possible to provide a cell culture device that can be more easily reproduced than a cell culture device using a conventional porous membrane.

It is a perspective view showing the whole cell culture device structure in a 1st embodiment. It is an exploded perspective view showing the whole cell culture device structure in a 1st embodiment. It is a schematic diagram which shows a part of BB line cross section in FIG. It is a top view which shows the porous membrane of the cell culture device in 1st Embodiment. 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. It is a schematic diagram which shows a part of DD cross section in FIG. 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. It is sectional drawing which shows the modification of the cell culture device in 3rd Embodiment. It is sectional drawing which shows the modification of the cell culture device in 3rd Embodiment. It is sectional drawing which shows the modification of the cell culture device in 3rd Embodiment. It is sectional drawing which shows another modification of the cell culture device in 3rd Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 4th Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 4th Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 4th Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 5th Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 5th Embodiment. It is sectional drawing which shows the principal part of the cell culture device in 5th Embodiment. It is sectional drawing which shows the example which laminates|stacks and uses a porous film. It is sectional drawing which shows the example which laminates|stacks and uses a porous film. It is sectional drawing which shows the example which laminates|stacks and uses a porous film. It is sectional drawing which shows the example which laminates|stacks and uses a porous film. It is a top view photograph of the porous membrane of an example. It is a top view photograph of the porous membrane of an example. It is a top view photograph of the porous membrane of a comparative example. It is a schematic cross section of the porous membrane of a comparative example. The relationship between the tensile force and the elongation of the porous membranes of Examples and Comparative Examples is shown in a graph. The relationship between the tensile force and the elongation of the porous membranes of Examples and Comparative Examples is shown in a graph.

Hereinafter, each embodiment of the present disclosure will be described with reference to the drawings. It should be noted that the following embodiments exemplify the present disclosure and do not limit the scope of the present disclosure. Further, in order to facilitate the description of each component, the dimensions of each component in the drawing are changed as appropriate. Therefore, the scale in the figure is different from the actual scale. In addition, some of the drawings are schematic diagrams in which each configuration is simplified. Further, in some drawings, description of common components represented by the same reference numeral may be omitted.

[First Embodiment]
<Cavity unit>
As shown in FIGS. 1 and 2, 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.

As shown in FIG. 2, on the upper surface of the lower cavity member 14, that is, the surface 14A facing the upper cavity member 12, there is formed a recess 26 that defines the lower microchannel 24 as one of the microcavities. There is. 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.

Similarly, on the lower surface of the upper cavity member 12, that is, the surface 12A facing the lower cavity member 14, there is formed a recess 20 that defines the upper microchannel 18 as the other of the microcavities. 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. Further, 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). Specifically, 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. And 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. In the present embodiment, as shown in FIG. 3 schematically showing a cross section taken along the line BB of FIG. 1, 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.

Here, 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. On the other hand, 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.

Further, 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.

<Porous membrane>
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.

As the hydrophobic polymer, polystyrene, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyhexafluoropropene, polyvinyl ether, polyvinylcarbazole, polyvinyl acetate, polytetrachloride. Fluoroethylene, 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. Further, any block copolymer that can be dissolved in a hydrophobic organic solvent can be used. Specific examples of 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. ..

Specifically, 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.

As shown in FIGS. 4 and 5, 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. Further, as shown in FIG. 4, 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. Here, 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.

As shown in FIG. 5, 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. Further, 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. ..

Further, as in another example shown in FIG. 6, 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.

When the cell culture device 10 of the present embodiment is used as a cell culture device or the like, at least 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 .. By covering the porous film 30, it becomes possible to enhance the adhesiveness of cells.

When 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.

For example, as shown in FIG. 7, 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.

Further, as shown in FIG. 9, 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. In this case, in order to prevent the cells constituting the cell layer 100 on each surface from passing through the porous membrane 30 and causing contamination with each other, it is desirable that the diameter of the openings 32A be 2 to 5 μm, which is smaller than the diameter of the cells. In this case, the film thickness is preferably 2 to 20 μm.

Further, as shown in FIG. 10, the cell layer 100 can be provided in the inner space 32 of the porous membrane 30. In this case, the porous film 30 in which the intra-membrane space 32 is open only on one surface is desirable. In this case, the film thickness is preferably 20 to 200 μm. When the cell layer 100 is provided in the intra-membrane space 32 of the porous membrane 30 as described above, the porous membrane 30 having the opening 32A provided on only one surface as shown in FIG. 6 may be used.

As described above, 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. ..

As a method for producing the porous film 30 in which the intra-membrane space 32 is formed, there is a condensation method, for example. 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 μm 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. Details of the dew condensation method are described in, for example, Japanese Patent Nos. 4945281, 5422230, 2011-74140, and 5405374, the contents of which are included in the present disclosure. Here, the opening ratio can be defined as the ratio of the area of the openings 32A to the area of the porous film 30. As 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.

In the present embodiment, the average opening diameter of the openings 32A is 1 μm or more and 200 μm or less. Here, since the average opening diameter of the openings 32A is 1 μm or more, it is easy to form the lateral communication structure of the intramembrane space 32. Further, since the average opening diameter of the openings 32A is 200 μm 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 μm or more and 200 μm 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.

In this embodiment, the porosity of the porous film 30 is 40% or more and 90% or less. Here, since 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. Further, since 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.

In the present embodiment, the film thickness of the porous film 30 is 0.5 μm or more and 100 μm or less. Here, 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. When the single layer porous film 30 is used, the film thickness is preferably 0.5 to 10 μm. When a plurality of porous films 30 are laminated and used, the total film thickness of the porous films 30 is preferably 10 to 200 μm.

In the present embodiment, 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.

<Holding member>
As shown in FIGS. 1 and 2, 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.

As shown in FIG. 2, 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. Further, 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.

As shown in FIG. 1,

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. Similarly, 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.

In addition to the accelerating mechanism 70, for example, as the film stretching mechanism, 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. Alternatively, a pressure may be applied. Further, 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.

As shown in FIG. 11, 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.

The mechanical stress applied to cells will be described with reference to FIGS. 12 to 14. 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. In addition, 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. In FIG. 12, 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.

Here, if the flow velocity of the liquid flowing through the lower micro flow channel 24 is controlled to be higher than the flow velocity of the liquid flowing through the upper micro flow channel 18 by the acceleration mechanism 70, the pressure in the thickness direction also changes due to the change in the flow velocity. Change. Here, when 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. In this case, as shown in FIG. 13, 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.

On the other hand, 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. When the pressure becomes higher than the pressure in the flow channel 24, as shown in FIG. 14, 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.

As described above, in the present embodiment, due to the extension characteristics based on the material and specifications of the porous film 30, 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.

By intermittently changing the flow velocity by the

acceleration mechanisms

70 and 75, it is possible to generate a pulse-like mechanical stress in the porous film 30.

Further, by connecting a device for pressurizing a liquid to either one or both of the upper microchannel 18 and the lower microchannel 24, one or both of the upper microchannel 18 and the lower microchannel 24 are connected. It is also possible to pressurize the liquid to expand the porous film 30.

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. .. For example, when intestinal epithelial cells are seeded on the porous membrane 30 as cells, it is possible to simulate the peristaltic movement of the intestinal tract by repeating expansion and contraction of the porous membrane 30, thereby forming an intestinal epithelial structure such as a crypt. Further, when cardiomyocytes are seeded on the porous membrane 30 as cells, mechanical stress on the porous membrane 30 can promote autonomous pulsation of the cardiomyocytes.

[Second Embodiment]
As shown in FIGS. 15 and 16, 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.

As shown in FIG. 16, on the upper surface of the lower cavity member 14, that is, the surface 14A facing the upper cavity member 12, a recess 26 that defines the lower microchannel 24 as one of the microcavities is formed. There is. 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.

As the microcavity in this embodiment, 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. In the present 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. In FIG. 17, 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. On the other hand, 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.

As shown in FIG. 18, which schematically shows a cross section taken along the line DD of FIG. 15, 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. By filling, 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. Further, as shown in FIG. 19, 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. Thereby, for example, the cardiomyocytes can be seeded on the porous membrane 30 to simulate the electrical dynamics of the cardiomyocytes. Note that 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. In addition, 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.

By using this acceleration mechanism 70, shear stress can be applied to the cell layer 100 from the lower microchannel 24 through the porous membrane 30 as in the first embodiment. 20 and 21 show a cross section taken along the line EE of FIG. 15, omitting configurations other than the upper cavity member 12 and the lower cavity member 14, the well 19, the lower microchannel 24, and the porous membrane 30. There is.

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. Here, when 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.

As described above, also in the present embodiment, due to the extension characteristics based on the material and specifications of the porous film 30, 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.

By intermittently changing the flow velocity by the acceleration mechanism 70, pulsed mechanical stress can be generated in the porous film 30.

Further, by connecting a device that pressurizes the liquid to the lower microchannel 24, it is possible to pressurize the liquid in the lower microchannel 24 and expand the porous membrane 30.

[Third Embodiment]
As shown in FIGS. 22 and 23, 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.

Further, the 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.

Further, 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. To do. 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.

Note that, as shown in FIG. 22,

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

inflow tubes

outflow tubes

62B and 64B. And cell suspension etc. flow out.

In the present embodiment, 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.

As shown in FIG. 24, when the movable member 85 is not operating, the lower cavity member 14 is not deformed and the porous membrane 30 is not expanded.

Here, as shown in FIG. 25, 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.

On the other hand, as shown in FIG. 26, 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.

Note that 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.

As shown in FIG. 27, when the movable member 85 is not operating, the upper cavity member 12 is not deformed and the porous membrane 30 is not expanded.

Here, as shown in FIG. 28, 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.

On the other hand, as shown in FIG. 29, 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.

Here, as shown in FIG. 30, 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.

By intermittently changing the flow velocity by the contact member 80 and the movable member 85, it is possible to generate a pulse-like mechanical stress in the porous film 30.

[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.

In the present embodiment, as shown in FIG. 31, 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.

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.

On the other hand, when gas is sucked from the internal pressure change space 90 by a vacuum pump as a pressurizing/depressurizing mechanism to reduce the pressure, 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. In this case, 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. By being applied as a pressing force to the membrane 30, the porous membrane 30 extends downward along the thickness direction. Further, 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. When applied as a traction force to 30, 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. Means that

[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.

In the present embodiment, as shown in FIG. 34, 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. Further, 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. .

When the gas is sucked from the side pressure change space 95B by a vacuum pump as a pressurization mechanism to reduce the pressure, the side pressure change space 95B contracts, as shown in FIG. Due to this contraction, the cavity side walls 95A are respectively pulled outward, and accordingly, the porous film 30 extends in the width direction. Thereby, mechanical stress in the width direction is applied to the porous film 30. 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.

On the other hand, when gas is sent to the side pressure change space 95B from a pressurizing pump as a pressurization mechanism to pressurize the side pressure change space 95B, the side pressure change space 95B expands inward as shown in FIG. By this expansion, the cavity side walls 95A are pressed inward, respectively, and the porous film 30 bends downward along the thickness direction. As a result, mechanical stress is applied to the porous film 30 in the width direction and the thickness direction.

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. Means that

[Lamination of porous film]
In each of the above embodiments, the porous film 30 has been used as a single layer, but a plurality of porous films 30 may be laminated and used. For example, as shown in 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.

Note that, as shown in FIG. 38, when two porous films 30 having openings 32A having the same diameter and having the same thickness are stacked and used with the positions of the openings 32A shifted, the porosity is increased. The aperture ratio can be substantially reduced without changing. Here, 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. By stacking the porous membranes 30 in this manner, it is possible to control the passage of cells through the porous membranes 30 while using the porous membranes 30 having the openings 32A larger than the cell diameter.

Further, as shown in FIG. 39, three or more porous membranes 30 having openings 32A having the same diameter and having the same thickness can be laminated. Furthermore, as shown in 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.

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 μm, the opening diameter was 5 μm, the porosity was 85%, and the opening ratio was 63%. Further, three layers of PB-made porous membranes were used as Example 3, and three layers of PC-made porous membranes were used as Example 4. These three-layer laminated porous films are as shown in the plan photograph of FIG. 42, and the aperture ratio is 11%. As is clear from FIG. 42, even if the same porous film is laminated, the position of the opening is displaced, so that the aperture ratio can be reduced. Therefore, even if a porous film having a small opening diameter is not prepared, the aperture ratio of the porous film can be substantially reduced by stacking the porous films having larger diameter openings. This makes it possible to prevent cells from slipping through the porous membrane even when culturing cells having a diameter larger than the diameter of the openings.

On the other hand, 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 μm, the opening diameter is 5 μm, the porosity is 10%, and the opening ratio is 3 to 10%.

For each of the examples and comparative examples, the respective materials, modes of lamination, and tensile force per unit width required for 20% elongation are shown in Table 1 below.

As shown in Table 1 above, 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). When 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%. Further, in Examples 1 and 3 in which PB is used as a material, the tensile force per unit width required for 20% extension is 6 as compared with Examples 2 and 4 in which the lamination mode is the same. It was possible to stretch with a smaller force of 4%.

Further, regarding the porous membranes of Examples 1 and 2 and Comparative Example, the relationship between the tensile force applied to the porous membrane per width and the elongation of the porous membrane is shown in the graphs of FIGS. 45 and 46. Note that 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). In the porous membrane of Example 2 made of the same material as the comparative example, 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). On the other hand, 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.

Claims

A pair of cavity members facing each other, each having a microcavity formed on the facing surface,
A porous membrane having a plurality of openings arranged between the facing surfaces of a pair of the cavity members and separating each of the microcavities and having one side or both sides arranged in a honeycomb shape,
A membrane stretching mechanism, which is connected to the cavity member or the porous membrane and can apply a force along at least the thickness direction to the porous membrane to stretch the porous membrane at least along the thickness direction,
Equipped with
The porous membrane has a plurality of intra-membrane spaces communicating from each of the plurality of openings in the thickness direction, and the adjacent intra-membrane spaces have a lateral communication structure in which they communicate with each other inside the porous membrane. Cage,
The average opening diameter of the openings is 1 μm or more and 200 μm or less,
The porosity of the porous film is 40% or more and 90% or less,
The thickness of the porous film is 0.5 μm or more and 100 μm or less,
The tensile force per unit width required for 20% extension of the porous film is 0.1 N/m or more and 20 N/m or less, and
At least one of the microcavities is a microchannel,
Cell culture device.
The cell culture device according to claim 1, wherein the porous membrane has the plurality of openings on both sides, and the opening on one surface communicates with the opening on the other surface through the intramembrane space.
The cell culture device according to claim 1 or 2, wherein the other of the microcavities is a well having an opening on the side opposite to the porous membrane.
The cell culture device according to claim 1 or 2, wherein the other of the respective microcavities is also a microchannel.
The cell culture device according to any one of claims 1 to 4, wherein a plurality of the porous membranes having the same opening diameter or different opening diameters are laminated.
The cell culture device according to any one of claims 1 to 5, further comprising, as the membrane stretching mechanism, an acceleration mechanism capable of increasing a flow velocity of a liquid flowing through the microchannel in the microchannel. ..
Of the pair of cavity members, at least the one in which the microchannel is formed has flexibility,
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 at least presses the contact member along the thickness direction of the porous membrane. And a movable member capable of one of pulling, and the cell culture device according to any one of claims 1 to 5.
Of the pair of cavity members, at least the one in which the microchannel is formed has flexibility,
The membrane stretching mechanism applies pressure along the thickness direction of the porous membrane from the side opposite to the side where the micro-channel is formed, which is formed inside the cavity member where the micro-channel is formed. The cell culture device according to any one of claims 1 to 5, comprising an internal pressure change space capable of at least one of pressing by and depressurization.
In addition to the membrane stretching mechanism,
Flexible cavity side walls provided on both sides of the microcavity,
A lateral pressure change space provided on the outside of each of the cavity sidewalls;
Has a lateral extension mechanism with
The lateral extension mechanism performs at least one of pressurization and depressurization in the lateral pressure change space, thereby performing at least one of pressurization by pressurization and pulling by depressurization on the side wall of the cavity. The cell culture device according to claim 1, wherein the cell culture device is expanded in the width direction.