Cell culture device and cell culture method

Abstract

To provide novel cell culture devices and cell culture methods.SOLUTION: Provided is a cell culture device 1 comprising: a lower substrate 10 that includes a cell culture part 14 having a bottom membrane; and an upper substrate 12 that is provided above the lower substrate 10 and that includes a cell housing part 24 having a bottom membrane in which micropores are formed, wherein there is a predetermined interval in the vertical direction between the bottom membrane of the lower substrate 10 and the bottom membrane of the upper substrate 12 when the upper substrate 12 is placed on the lower substrate 10. Also provided is a cell culture method that uses the cell culture device 1.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture device and a cell culture method using such a cell culture device. [Background technology]

[0002] In the fields of medicine and drug discovery, cells have been cultured and analyzed in cell culture devices to analyze the structure and function of various cells, tissues, and organs in living organisms. Known analytical methods using such cell culture devices include, for example, a method in which a large number of minute cell culture sections are arrayed using microfabrication technology that applies semiconductor processing technology, and a single cell is cultured in each cell culture section, thereby analyzing single-cell level function with high throughput, a method in which multiple types of cells are co-cultured in minute cell culture sections formed using microfabrication technology, thereby analyzing intercellular communication, and a method in which tissues or organs reconstructed in minute cell culture sections are used to analyze pharmacokinetics, etc.

[0003] One cell culture device that can be used in such analysis methods is one in which the bottom of each well of a microwell array is constructed from a free-standing SiN membrane using semiconductor processing technology, with numerous micro through-holes with a diameter of approximately 3 μm formed in the SiN membrane (see Non-Patent Document 1). By culturing numerous astrocytes on the backside of this SiN membrane and single neurons on the surface of the SiN membrane in each well, intercellular communication through the micro through-holes is possible, and the physiological activity of single neurons has been successfully maintained for a long period of time.

[0004] However, when seeding cells directly onto a microwell array using a micropipette, the probability of successfully introducing a single cell into each well was low and extremely difficult. If the well dimensions could be reduced to nearly the size of a cell, it would be physically difficult to fit more than one cell into one well, and the success rate would be relatively high. However, when culturing adherent cells such as neurons, which require a large culture area, the well dimensions would inevitably be larger, making it difficult to introduce a single cell into each well, and the success rate was only a few percent.

[0005] Also, a two-layer microchannel device separated by a porous membrane fabricated using semiconductor processing technology has been proposed (see Patent Document 1). A retinal layer is formed in the first microchannel by culturing retinal pigment epithelial cells, and a vascular layer is formed in the second microchannel by culturing vascular endothelial cells and fibroblasts, thereby constructing a model of a blood-retinal barrier consisting of two layers.

[0006] However, to form tissues with three or more layers using such two-layer microfluidic devices separated by a porous membrane, it was necessary to install two or more porous membranes within the microchannel. Furthermore, such devices are fabricated by bonding upper and lower microchannels, which are fabricated using semiconductor processing technology, with porous membranes made of plastic materials such as polyethylene terephthalate (PET). The more layers added, the worse the alignment accuracy during bonding became. Furthermore, it was difficult to control the distance between layers. For example, even if cells in the first and second layers could be placed close to each other across the porous membrane, it was difficult to bring the cells in the second and third layers close enough together. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2020-188723 [Non-patent literature]

[0008] [Non-Patent Document 1] Yusuke Zenmyo, Isamu Morisako, Takashi Yasuda, Long-term culture of single neurons using microwells with SiN porous membranes, Transactions of the Institute of Electrical Engineers of Japan, Vol. 138, No. 7, pp. 327-328 (2018) Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel cell culture device and a culture method using such a cell culture device. [Means for solving the problem]

[0010] The present inventors have discovered a novel cell culture device useful for analyzing functions at the single cell level and for analyzing intercellular communication, and a culture method using the cell culture device, and have completed the present invention.

[0011] That is, the present invention is as follows. [1] A lower substrate having a cell culture area with a bottom membrane; an upper substrate disposed above the lower substrate and having a cell storage portion with a bottom membrane having micropores formed therein; A cell culture device characterized in that, when the upper substrate is disposed on the lower substrate, the bottom film of the lower substrate and the bottom film of the upper substrate are arranged vertically with a predetermined distance between them.

[0012] [2] The cell culture device according to [1] above, wherein one micropore is formed in the bottom membrane of the upper substrate. [3] The cell culture device according to [2] above, wherein a plurality of micropores are formed in the bottom membrane of the lower substrate. [4] The cell culture device according to [2] or [3] above, wherein the bottom film of the upper substrate and / or the lower substrate is made of a transparent material. [5] The cell culture device described in [4] above, wherein the bottom film of the upper substrate and / or lower substrate contains at least one inorganic material selected from silicon nitride (SiN), silicon oxide (SiO2), and silicon oxynitride (SiON). [6] The lower substrate has a convex portion or a concave portion, and the upper substrate has a concave portion or a convex portion; The cell culture device according to any one of [2] to [5] above, characterized in that the convex or concave portion of the lower substrate engages with the concave or convex portion of the upper substrate, thereby aligning the bottom membrane of the lower substrate with the bottom membrane of the upper substrate. [7] A cell culture device according to any one of [2] to [6] above, characterized in that the lower substrate comprises a plurality of cell culture sections arranged in a lattice pattern, and the upper substrate comprises a plurality of cell storage sections arranged in a lattice pattern. [8] The cell culture device described in [7] above, characterized in that the upper part of the lower partition member that separates the plurality of lattice-shaped cell culture sections of the lower substrate forms a convex part, and the lower part of the upper partition member that separates the plurality of cell storage sections of the upper substrate that correspond to the lower partition member has a concave part that corresponds to the upper convex part of the lower partition member.

[0013] [9] The cell culture device described in [1] above, characterized in that a plurality of micropores are formed in the bottom membrane of the cell culture section of the lower substrate, and a plurality of micropores are formed in the bottom membrane of the cell storage section of the upper substrate.

[10] The cell culture device according to [9] above, wherein the bottom film of the upper substrate and / or the lower substrate is made of a transparent material.

[11] The cell culture device described in

[10] above, wherein the bottom film of the upper substrate and / or lower substrate contains at least one inorganic material selected from silicon nitride (SiN), silicon oxide (SiO2), and silicon oxynitride (SiON).

[12] The cell culture device according to any one of [9] to

[11] above, characterized in that the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell storage section of the upper substrate are arranged with a gap of 5 to 500 μm between them.

[13] The lower substrate has a convex portion or a concave portion, and the upper substrate has a concave portion or a convex portion; The cell culture device according to any one of [9] to

[12] above, characterized in that the bottom membrane of the lower substrate is aligned with the bottom membrane of the upper substrate by engaging the convex or concave portion of the lower substrate with the concave or convex portion of the upper substrate.

[14] A cell culture device according to any one of [9] to

[13] above, characterized in that the lower substrate comprises a plurality of cell culture sections arranged in a lattice pattern, and the upper substrate comprises a plurality of cell storage sections arranged in a lattice pattern.

[15] The cell culture device described in

[14] above, characterized in that the upper part of the lower partition member that separates the plurality of lattice-shaped cell culture sections of the lower substrate forms a convex part, and the lower part of the upper partition member that separates the plurality of cell storage sections of the upper substrate that correspond to the lower partition member has a concave part that corresponds to the upper convex part of the lower partition member.

[16] The cell culture device according to any one of [1] to

[15] above, wherein the lower substrate and the upper substrate are fabricated using photolithography, which is a semiconductor microfabrication technology.

[0014]

[17] A cell culture method for culturing cells using the cell culture device according to any one of [2] to [8] and

[16] above, A cell culture method characterized by seeding cells on the bottom membrane of the cell storage section of the upper substrate, dropping the cells onto the bottom membrane of the cell culture section of the lower substrate through a single microhole formed in the bottom membrane of the cell storage section of the upper substrate, and culturing the cells in the cell culture section.

[18] A cell culture method for culturing cells using the cell culture device according to any one of [9] to

[16] above, A cell culture method comprising culturing cells on one or both sides of the cell culture membrane of the lower substrate and culturing cells on one or both sides of the cell culture membrane of the upper substrate. [Effects of the Invention]

[0015] According to the present invention, it is possible to provide a novel cell culture device and a cell culture method that are useful for analyzing functions at the single cell level, analyzing intercellular communication, and the like. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic explanatory view of a cell culture device according to an embodiment of the first invention. [Figure 2] 2 is a schematic cross-sectional view of a lower substrate and an upper substrate of the cell culture device of FIG. 1. FIG. [Figure 3] FIG. 2 is an explanatory diagram of a cell culture method using the cell culture device of FIG. [Figure 4] FIG. 10 is a schematic explanatory view of a cell culture device according to an embodiment of the second invention. [Figure 5] 5 is a schematic cross-sectional view of the lower substrate and the upper substrate of the cell culture device of FIG. 4. [Figure 6] FIG. 5 is an explanatory diagram of a cell culture method using the cell culture device of FIG. [Figure 7] 1 shows photographs of the cell culture device produced in the examples, where (a) shows the upper substrate and (b) shows the lower substrate. [Figure 8] FIG. 10 shows the results of a cell seeding test using a cell culture device prepared in an example, showing the "number of wells" based on the "number of cells per well" (e.g., the number of wells into which one cell was introduced). DETAILED DESCRIPTION OF THE INVENTION

[0017] The cell culture device of the present invention comprises a lower substrate having a cell culture section with a bottom membrane, and an upper substrate disposed above the lower substrate and having a cell storage section with a bottom membrane formed with micropores, wherein when the upper substrate is disposed on the lower substrate, the bottom membranes of the lower substrate and the upper substrate are disposed vertically with a predetermined distance between them. The cell culture device of the present invention is not limited to a two-layer arrangement of substrates, but may also be a stack of three or more layers.

[0018] The cell culture device of the present invention is useful for analyzing functions at the single cell level, analyzing intercellular communication, and the like.

[0019] The cells to be cultured in the cell culture device of the present invention are not particularly limited and can be selected appropriately depending on the purpose. Examples include cells that form tissues such as skin, retina, cardiac muscle, blood vessels, nerves, and organs extracted from humans or animals, cell lines derived from these cells, stem cells such as mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPS cells), and embryonic stem cells (ES cells), and cells that are induced to differentiate from stem cells and form tissues such as nerves, skin, cardiac muscle, and liver.

[0020] <Cell culture device according to the first invention> The cell culture device according to the first aspect of the present invention will be described below. The cell culture device of the first invention comprises a lower substrate having a cell culture section with a bottom membrane, and an upper substrate arranged above the lower substrate and having a cell storage section with a bottom membrane having one micropore formed therein, and is characterized in that when the upper substrate is arranged on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged vertically at a predetermined distance from each other.

[0021] The cell culture device according to the first invention allows a single cell to be seeded into the cell culture section of the lower substrate with a high probability by dropping the cell through a single microhole formed in the bottom membrane of the cell storage section of the upper substrate. In other words, compared to the case of directly seeding cells using a conventional micropipette, it is possible to introduce only one cell into one cell culture section (well) with a high probability. Furthermore, even when the area of ​​the cell culture section is large, it is possible to introduce a single cell with a high probability, making it suitable for use in culturing adherent cells such as neurons, which require a large culture area.

[0022] Each component of the cell culture device according to the first invention will be described below. [Lower board] The lower substrate is a substrate equipped with a cell culture area, and examples of the material thereof include ceramic wafers such as silicon wafers, glass wafers, quartz wafers, etc. The thickness is preferably 200 μm or more, more preferably 200 to 500 μm, and even more preferably 250 to 350 μm.

[0023] (Cell Culture Department) The cell culture section is a site where cells are seeded and cultured, and is, for example, a well (depression) formed in the lower substrate. The shape of the internal space of the cell culture section is not particularly limited, and examples thereof include a cylindrical shape, a prismatic shape, an inverted truncated cone shape, and an inverted truncated pyramid shape, with an inverted truncated pyramid shape being preferred, and an inverted truncated square pyramid shape being particularly preferred. One or more cell culture sections may be provided, and the number thereof generally corresponds to the number of cell storage sections in the upper substrate described below, and they are arranged at positions corresponding to the cell storage sections in the upper substrate. It is preferable that a large number of cell culture sections are arranged in a lattice pattern (array pattern).

[0024] When the cell culture sections are arranged in a lattice pattern, the cell culture sections are partitioned and divided by partition members (lower partition members) arranged in a lattice pattern that form the side walls (peripheral walls) of the cell culture sections. It is preferable to align the cell culture sections of the lower substrate and the cell storage sections of the upper substrate by fitting a recess formed in the lower part of the upper partition member of the cell storage section of the upper substrate, which will be described later, into all or part of the upper part of this lower partition member.

[0025] The size (diameter (diagonal in the case of a polygon)) of the bottom membrane (cell culture membrane) of the cell culture part is preferably 100 to 1000 μm, more preferably 150 to 800 μm, and even more preferably 200 to 600 μm.

[0026] The cell culture membrane does not necessarily have to have micropores, but preferably has multiple micropores. This allows, for example, when cells are supported on one side of the cell culture membrane, nutrients from the culture medium can be supplied from the other side through the micropores. Furthermore, when cells are supported on both sides of the cell culture membrane, intercellular communication can be smoothly achieved through the micropores.

[0027] The shape of the micropores can be appropriately determined depending on the type of cells, the purpose of culture, etc., and examples thereof include circular and polygonal shapes. The diameter of the micropores (diagonal in the case of polygons) is preferably large enough to prevent cells from passing through, and is preferably 20 μm or less, more preferably 0.1 to 10 μm, even more preferably 0.5 to 5 μm, and particularly preferably 1 to 4 μm. The area ratio of the micropores in the bottom membrane of the cell culture section varies depending on the diameter of the micropores, but is preferably 10 to 70%, more preferably 10 to 50%, and even more preferably 10 to 20%. Within this range, nutrients can be smoothly supplied through the micropores and intercellular communication can be smoothly achieved, and in the case of transparent materials, high transparency can be maintained.

[0028] The thickness of the cell culture membrane is, for example, preferably 0.1 to 5 μm, more preferably 0.5 to 1.5 μm, in which case the membrane can maintain its mechanical strength as a scaffold for cells and, in the case of a transparent material, maintain high transparency.

[0029] The material of the cell culture membrane is not particularly limited as long as it can serve as a scaffold for cells. However, a transparent material is preferred for ease of cell observation. Transparent materials may be organic materials, but inorganic materials are preferred. Examples of inorganic materials include transparent nitrides, oxides, and oxynitrides of silicon, titanium, zinc, tin, and aluminum. Specific examples include silicon nitride (SiN), silicon oxide (SiO), titanium oxide (TiO), zinc oxide (ZnO), tin oxide (SnO), silicon oxynitride (SiON), titanium oxynitride (TiON), indium oxide (InO), and indium tin oxide (ITO). Silicon nitride, silicon oxide, and silicon oxynitride are preferred, with silicon nitride or silicon oxide being particularly preferred. Two or more of these transparent inorganic materials may be used in combination. Alternatively, a laminated film of two or more types may be formed. For example, the surface of an inorganic material may be coated with a transparent organic material. In this specification, the term "transparent" means that the light transmittance at wavelengths of 500 nm to 600 nm is 70% or more, preferably 80% or more, and more preferably 90% or more.

[0030] The surface of the bottom membrane of the cell culture section (cell culture membrane) is preferably modified with functional molecules to promote cell adhesion and cell proliferation. The functional molecules are not particularly limited as long as they can be used for cell culture, and examples include extracellular matrices such as collagen, proteoglycan, heparan sulfate proteoglycan, fibronectin, laminin, entactin, elastin, hyaluronic acid, and tenascin; cell adhesion peptides such as arginine-glycine-aspartic acid, leucine-aspartic acid-valine, and arginine-glutamic acid-aspartic acid-valine; and synthetic molecules such as poly-L-lysine and poly-L-ornithine.

[0031] (Support frame part) The lower substrate preferably has a support frame (holder) that supports the lower substrate. This allows the upper substrate to be more stably positioned on the lower substrate. It can also be used as a grip, improving operability. Examples of materials for the support frame include synthetic resins.

[0032] [Upper board] The upper substrate is disposed above the lower substrate and is provided with a cell-containing section having a bottom membrane with one micropore formed therein. The material of the substrate can be the same as that of the lower substrate, and may be the same material as or a different material from that of the lower substrate. The thickness of the upper substrate is preferably 200 μm or more, more preferably 200 to 500 μm, and even more preferably 250 to 350 μm.

[0033] (Cell storage section) The cell storage section is a section capable of storing cells, and is, for example, a well (depression) formed in the upper substrate. The shape of the internal space of the cell storage section is not particularly limited, and examples thereof include a cylindrical shape, a prismatic shape, an inverted truncated cone shape, and an inverted truncated pyramid shape, with an inverted truncated pyramid shape being preferred, and an inverted truncated square pyramid shape being more preferred. One or more cell storage sections may be provided, and typically the number corresponds to the number of cell culture sections in the lower substrate, and they are arranged at positions corresponding to the cell culture sections in the lower substrate. It is preferable that multiple cell storage sections are arranged in a lattice pattern (array). When two or more cell storage sections are provided, it is preferable that each cell storage section has the same configuration so that cells fall evenly.

[0034] When the cell holding sections are arranged in a lattice pattern, the cell holding sections are partitioned and divided by partition members (upper partition members) arranged in a lattice pattern that form the side walls (peripheral walls) of the cell holding sections. As described above, it is preferable to form a recess in the lower part of this upper partition member and fit a protrusion in the upper part of the lower partition member of the cell culture section of the lower substrate, thereby aligning the two. Usually, when the upper substrate is placed on the lower substrate, a portion of the cell holding section of the upper substrate is introduced into the cell culture section of the lower substrate. Therefore, the bottom membrane of the cell holding section of the upper substrate is smaller than the bottom membrane of the cell culture section of the lower substrate, preferably about 1 / 3 to 2 / 3.

[0035] The bottom membrane of the cell-containing section has a single micropore large enough to allow cells to pass through. This single micropore is preferably formed in the center of the bottom membrane of the cell-containing section. This allows the micropore to be positioned above the center of the bottom membrane of the cell culture section of the lower substrate when the upper substrate is placed above the lower substrate, ensuring reliable cell drop into the cell culture section of the lower substrate. The shape of the micropore can be appropriately determined depending on the type of cell, and examples include circular and polygonal shapes. The diameter of the micropore (diagonal in the case of a polygon) can be appropriately determined depending on the type of cell, and is preferably at least the diameter of one cell but less than the sum of the diameters of two cells. For example, a diameter of 3 to 50 μm is preferred, and a diameter of 8 to 20 μm is more preferred. For example, in the case of neurons, the diameter is approximately 8 μm, so a micropore with a diameter of 10 to 15 μm is preferred. This allows for a high probability of seeding only one cell per cell culture section.

[0036] (Support frame part) The upper substrate preferably has a support frame (holder) that supports the upper substrate. This allows the upper substrate to be more stably positioned on the lower substrate. It can also be used as a grip, improving operability. Examples of materials for the peripheral wall include synthetic resins.

[0037] [Alignment of the cell culture section of the lower substrate and the cell storage section of the upper substrate] In the cell culture device according to the first invention, when the upper substrate is disposed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are vertically spaced apart at a predetermined distance, and one microhole in the bottom membrane of the upper substrate is positioned above the bottom membrane of the lower substrate. In this case, it is preferable that the centers of the bottom membranes are aligned. This allows cells to be dropped reliably and accurately onto the bottom membrane of the cell culture section of the lower substrate. The distance between the bottom membranes is preferably 5 to 500 μm, more preferably 20 to 250 μm, and even more preferably 50 to 200 μm.

[0038] The manner in which the lower substrate and the upper substrate are aligned is not particularly limited, and examples include a manner in which concave and convex portions formed on the lower substrate and the upper substrate are fitted together, a manner in which peripheral convex portions of large and small diameters (diagonals in the case of polygons) formed on the outer peripheries of the lower substrate and the upper substrate are fitted together adjacent to each other, a manner in which the upper substrate is placed within the peripheral frame of the lower substrate, a manner in which marks on the lower substrate and the upper substrate are aligned and fixed with a fixing device, and a manner in which a through-hole formed in the lower substrate and the upper substrate are inserted and fixed.

[0039] Specifically, examples of embodiments in which the recesses and protrusions formed on the lower and upper substrates are fitted together include a lower substrate having a protrusion or recess and an upper substrate having a recess or protrusion, and the protrusions or recesses of the lower substrate and the recesses or protrusions of the upper substrate are fitted together to align the cell culture section of the lower substrate with the cell storage section of the upper substrate. More specifically, examples include a peripheral recess or peripheral protrusion surrounding the upper substrate being fitted together with a peripheral protrusion or peripheral recess surrounding the lower substrate, and an embodiment in which the upper part of a lower partition member separating the plurality of lattice-shaped cell culture sections of the lower substrate constitutes a protrusion, and the lower part of the upper partition member separating the plurality of cell storage sections of the upper substrate corresponding to the lower partition member is formed with a recess corresponding to the upper protrusion of the lower partition member, thereby fitting the lattice-shaped protrusion of the lower substrate with the lattice-shaped recess of the upper substrate. The lower substrate and the upper substrate may each have one protrusion or recess, or multiple protrusions or recesses.

[0040] An example of a mode in which peripheral convex portions formed on the lower substrate and the upper substrate are fitted adjacent to each other is a mode in which a peripheral convex portion surrounding the periphery of the lower substrate is fitted adjacent to a peripheral convex portion surrounding the upper substrate and having a diameter (diagonal in the case of a polygon) that is one size larger or smaller than the convex portion of the lower substrate.

[0041] In this manner of aligning the lower substrate and the upper substrate, the horizontal and vertical alignment (the distance between the cell culture membrane of the upper substrate and the cell storage portion of the lower substrate) of the cell culture section of the lower substrate and the cell storage portion of the upper substrate can be easily and accurately performed without using any special equipment, thereby improving operability during culture.

[0042] <Method for manufacturing the cell culture device according to the first invention> Next, a method for producing the cell culture device according to the first invention will be described. The method for producing the lower and upper substrates in the cell culture device according to the first invention is not particularly limited and can be various methods, but it is preferable to use photolithography, which is a semiconductor microfabrication technology, for example, a method similar to that for the cell culture sheet described in JP 2014-147342 A.

[0043] An example of a method for producing the cell culture device according to the first aspect of the present invention will be described below. Specifically, the cell culture device of the present invention will be described in terms of a case in which a lower substrate has a plurality of cell culture sections arranged in a lattice pattern, an upper substrate has a plurality of cell storage sections arranged in a lattice pattern, the upper part of a lower partition member separating the plurality of lattice-shaped cell culture sections of the lower substrate forms a convex portion, and a lower part of an upper partition member separating the plurality of cell storage sections of the upper substrate corresponding to the lower partition member has a concave portion corresponding to the upper convex portion of the lower partition member.Furthermore, the case in which silicon substrates having a (100) crystal orientation and a mirror surface on one side are used as the lower substrate and the upper substrate will be described.

[0044] (Preparation of the lower substrate) The lower substrate preparation step includes a cell culture section formation step and a bottom membrane micropore formation step. The cell culture area formation process may include, for example, the steps of forming a silicon nitride film (inorganic material film) as a transparent inorganic material on both sides of a silicon substrate using a chemical vapor deposition method such as plasma CVD, applying a photoresist (photosensitive resin) to the silicon nitride film on one side (non-mirror surface) of the silicon substrate to form a resist layer, irradiating the surface of the resist layer with ultraviolet light through a photomask with a pattern for multiple etching windows, immersing the silicon substrate in a developer to develop the resist layer and patterning the etching windows, etching the exposed silicon nitride film using a plasma-converted reactive gas using the remaining resist layer as a mask (protective film), and then removing the resist layer with an organic solvent, and immersing the silicon substrate in an etching solution to etch the silicon into a quadrangular truncated pyramid shape (inverted pyramid shape) along the silicon crystal planes to form multiple lattice-shaped cell culture areas. Note that lattice-shaped protrusions (peripheral walls of the cell culture areas) are formed simultaneously with the formation of the cell culture areas.

[0045] The bottom membrane micropore forming process may include the steps of applying photoresist (photosensitive resin) to the silicon nitride film on the other side (mirror surface) of the silicon substrate to form a resist layer, irradiating ultraviolet light through a glass mask with multiple micropores drawn on it per cell culture section, immersing the silicon substrate in a developer to develop the resist layer and pattern the micropores, and using the remaining resist layer as a mask (protective film) to etch the exposed silicon nitride film using plasmatized reactive gas to form micropores, and then removing the resist layer.

[0046] It is also preferable to have a final finishing step of immersing the silicon substrate in an etching solution to etch away the remaining silicon from the etching windows and the micropores along the crystal planes of the silicon.

[0047] (Preparation of upper substrate) The upper substrate preparation step includes a cell-receiving section formation step and a bottom micropore and recess formation step.

[0048] The cell holding section formation process includes, for example, the steps of forming a silicon nitride film (inorganic material film) as a transparent inorganic material on both sides of a silicon substrate using a chemical vapor deposition method such as plasma CVD, applying a photoresist (photosensitive resin) to the silicon nitride film on one side (non-mirror surface) of the silicon substrate to form a resist layer, irradiating the surface of the resist layer with ultraviolet light through a photomask with a rectangular pattern for multiple etching windows, immersing the silicon substrate in a developer to develop the resist layer and patterning the etching windows, etching the exposed silicon nitride film with a plasma-converted reactive gas using the remaining resist layer as a mask (protective film), and then removing the resist layer with an organic solvent, and immersing the silicon substrate in an etching solution to etch the silicon from the etching windows along the crystal planes of the silicon into a quadrangular truncated pyramid shape (inverted pyramid shape), forming multiple cell holding sections arranged in a lattice pattern. As with the lower substrate, lattice-shaped protrusions (peripheral walls of the cell holding sections) are formed simultaneously with the formation of the cell holding sections.

[0049] In addition, the bottom membrane micropore and recess formation process includes the steps of applying photoresist (photosensitive resin) to the silicon nitride film on the other side (mirror surface) of the silicon substrate to form a resist layer, irradiating ultraviolet light through a photomask on which one micropore and recess pattern per cell storage section is drawn, immersing the silicon substrate in a developer to develop the resist layer and pattern the micropores and recesses, using the remaining resist layer as a mask (protective film) to etch the exposed silicon nitride film using plasma-converted reactive gas to form the areas where the micropores and recesses are to be formed, and then removing the resist layer with an organic solvent, and immersing the silicon substrate in an etching solution to which a surfactant has been added to etch the silicon along the silicon crystal planes to form the recesses.

[0050] It is preferable to carry out a finishing process of etching away the silicon remaining in the cell-receiving section formation process simultaneously with the process of forming the recess.

[0051] <Cell culture method according to the first aspect of the present invention> Next, a cell culture method using the cell culture device according to the first aspect of the present invention will be described. The cell culture method according to the first invention is a cell culture method for culturing cells using the cell culture device according to the first invention, characterized in that cells are seeded on the bottom membrane of the cell storage section of the upper substrate, dropped onto the bottom membrane of the cell culture section of the lower substrate through a single microhole formed in the bottom membrane of the cell storage section of the upper substrate, and cultured in the cell culture section.

[0052] According to the cell culture method of the first invention, cells are seeded by dropping them into the cell culture section of the lower substrate through a single microhole formed in the bottom membrane of the cell storage section of the upper substrate, which allows for a higher probability of introducing a single cell into each well than the conventional method of directly seeding cells into a microwell array using a micropipette. Furthermore, although adherent cells such as neurons require a large culture area, this method allows for a high probability of introducing a single cell into such a large cell culture section.

[0053] Hereinafter, embodiments of the cell culture device according to one embodiment of the first invention will be specifically described with reference to the drawings, but the present invention is not limited to these embodiments.

[0054] Here, Fig. 1 is a schematic explanatory diagram of a cell culture device according to one embodiment of the first invention. Fig. 2 is a schematic cross-sectional view of a lower substrate and an upper substrate of the cell culture device of Fig. 1. Fig. 3 is an explanatory diagram of a cell culture method using the cell culture device of Fig. 1.

[0055] As shown in FIG. 1, a cell culture device 1 according to an embodiment of the first invention includes a square lower substrate 10 and a square upper substrate 12 disposed above the lower substrate 10.

[0056] As shown in Figures 2 and 3, the lower substrate 10 is provided with cell culture sections 14 arranged in a 4x4 grid pattern and having a bottom membrane with 16 300 µm squares (X). The bottom membrane of the cell culture sections 14 is a cell culture membrane made of silicon nitride and has approximately 200 micropores 18 with a diameter of approximately 3 µm. The upper part of the lower partition member 16 that separates the grid-like cell culture sections 14 of the lower substrate 10 forms a convex portion 20. The lower substrate 10 is provided with a PC (polycarbonate) holder 22 as a support frame that supports the lower substrate 10.

[0057] As shown in Figures 2 and 3, the upper substrate 12 is provided with cell storage sections 24 arranged in a 4x4 grid, each having a bottom membrane with 16 sections, each 150 µm square (Y). The bottom membrane of the cell storage section 24 is a silicon nitride membrane with a single microhole 26 approximately 12 µm in diameter formed in its center. A recess 30 corresponding to the upper protrusion 20 of the lower partition member 16 is formed in the lower part of the upper partition member 28 that separates the cell storage section 24 of the upper substrate 12. The distance (Z) between the bottom of the cell culture section 14 of the lower substrate 10 and the cell storage section 24 of the upper substrate 12 is approximately 150 µm, and horizontal and vertical alignment is achieved by the engagement of the upper protrusion 20 of the lower substrate 10 with the recess 30 of the upper substrate 12. The upper substrate 12 is provided with a PDMS (polydimethylsiloxane) frame 32 as a support frame for supporting the upper substrate 12.

[0058] Next, an example of a cell culture method using the above-described cell culture device 1 will be described. As shown in Figure 3, first, the upper surface of the cell culture section 14 of the lower substrate 10 is modified with functional molecules, while the lower surface of the cell culture section 14 of the lower substrate 10 is modified with functional molecules and astrocytes A are seeded (Figure 3(a)).

[0059] Next, the upper substrate 12 is superimposed on the lower substrate 10 (FIG. 3(b)). At this time, the convex portions 20 of the lower substrate 10 are fitted into the concave portions 30 of the upper substrate 12, and the top surfaces of the convex portions 20 of the lower substrate 10 are brought into contact with the bottom surfaces of the concave portions 30 of the upper substrate 12, thereby achieving horizontal and vertical alignment.

[0060] Next, a cell suspension of neurons is introduced into the cell holding section 24 of the upper substrate 12, and neuron B is seeded (FIG. 3(c)). This allows for a high probability of introducing one neuron B into the cell culture section 14 of the lower substrate 10 through the micropores 26 in the bottom membrane of the cell holding section 24.

[0061] Subsequently, the cell culture device 1 is placed in an incubator, and neurons B that have passed through the micropores 26 of the upper substrate 12 and reached the lower substrate 10 are allowed to adhere to the surface of the cell culture section 14 of the lower substrate 10. Subsequently, the upper substrate 12 is removed, and the culture is continued on the lower substrate 10 (FIG. 3(d)).

[0062] As described above, by using the cell culture device of the first invention, a single cell can be seeded with a high probability into the cell culture section of the lower substrate by dropping the cell through a single microhole in the cell storage section of the upper substrate. Furthermore, a single neuron B and an astrocyte A are co-cultured across the bottom membrane (cell culture membrane) of the cell culture section 14 of the lower substrate 10, and intercellular communication between the two cells is achieved through the microhole 18. This enables long-term culture of a single neuron, enabling analysis and drug efficacy evaluation at the single neuron level.

[0063] <Cell culture device according to the second invention> Next, a cell culture device according to a second aspect of the present invention will be described. The cell culture device of the second invention comprises a lower substrate having a cell culture section with a bottom membrane (cell culture membrane) having a plurality of micropores formed therein, and an upper substrate arranged above the lower substrate and having a cell storage section with a bottom membrane (cell culture membrane) having a plurality of micropores formed therein, characterized in that when the upper substrate is arranged on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged vertically at a predetermined distance from each other.

[0064] In the cell culture device according to the second invention, the cell culture membranes of the lower substrate and the upper substrate are arranged vertically at a predetermined interval, so that cells can be cultured in advance on one or both sides of the cell culture membranes of the upper and lower substrates, and then these substrates can be stacked to achieve good intercellular communication. Furthermore, it is possible to easily stack three or more substrates and perform cell culture.

[0065] The lower substrate of the cell culture device according to the second invention is the same as the lower substrate of the cell culture device according to the first invention (when a plurality of micropores is formed in the bottom membrane of the cell culture section). The upper substrate of the cell culture device according to the second invention is the same as the upper substrate of the cell culture device according to the first invention, except that the bottom membrane is the bottom membrane of the lower substrate of the cell culture device according to the first invention in which a plurality of micropores is formed. Furthermore, the manufacturing method of the cell culture device according to the second invention is almost the same as the manufacturing method of the cell culture device according to the first invention, and changes in the manufacturing method based on changes in the configuration of the device can be applied by appropriately applying the manufacturing method of the first invention.

[0066] <Cell culture method according to the second invention> Next, a cell culture method using the cell culture device of the second invention will be described. The cell culture method according to the second invention is a cell culture method for culturing cells using the cell culture device of the second invention, characterized in that cells are cultured on one or both sides of the cell culture membrane of the lower substrate, and cells are cultured on one or both sides of the cell culture membrane of the upper substrate.

[0067] According to the cell culture method of the second invention, cells are pre-cultured on one or both sides of the cell culture membranes of the upper and lower substrates, and a cell culture device is used in which the upper substrate is placed on the lower substrate, making it easy to culture cells in three or four layers.

[0068] Hereinafter, embodiments of the cell culture device according to one embodiment of the second invention will be specifically described with reference to the drawings, but the present invention is not limited to these embodiments.

[0069] Here, Fig. 4 is a schematic explanatory diagram of a cell culture device according to one embodiment of the second invention. Fig. 5 is a schematic cross-sectional diagram of the lower substrate and the upper substrate of the cell culture device of Fig. 4. Fig. 6 is an explanatory diagram of a cell culture method using the cell culture device of Fig. 4.

[0070] As shown in FIG. 4, a cell culture device 2 according to an embodiment of the second invention includes a square lower substrate 34 and a square upper substrate 36 disposed above the lower substrate 34.

[0071] As shown in Figures 5 and 6, the lower substrate 34 is provided with cell culture sections 38 arranged in a 4x4 grid pattern and having a bottom membrane with 16 300 µm squares (X). The bottom membrane of the cell culture sections 38 is a cell culture membrane made of silicon nitride and has approximately 200 micropores 40 with a diameter of approximately 3 µm. The upper part of the lower partition members 42 that separate the grid-like cell culture sections 38 of the lower substrate 34 constitutes a convex portion 44. The lower substrate 34 is provided with a PC (polycarbonate) holder 46 as a support frame that supports the lower substrate 34.

[0072] As shown in Figures 5 and 6, the upper substrate 36 is equipped with cell culture sections (cell storage sections) 48 arranged in a 4x4 grid and having 16 bottom membranes each 150 µm square (Y). The cell culture sections 48 are composed of a silicon nitride cell culture membrane with approximately 200 micropores 50 with a diameter of approximately 3 µm. The upper partition member 52 separating the 16 cell culture sections 48 of the upper substrate 36 has a recessed section 54 formed at the bottom thereof corresponding to the upper convex section 44 of the lower partition member 42. The distance (Z) between the bottom membrane of the cell culture section 38 of the lower substrate 34 and the bottom membrane of the cell culture section 48 of the upper substrate 36 is approximately 150 µm. The upper convex section 44 of the lower substrate 34 engages with the recessed section 54 of the upper substrate 36, ensuring horizontal and vertical alignment. The upper substrate 36 is equipped with a PDMS (polydimethylsiloxane) frame 56 as a support frame for supporting the upper substrate 36.

[0073] Next, an example of a cell culture method using the above-described cell culture device 2 will be described. First, the upper surface of the bottom membrane of the cell culture section 38 of the lower substrate 34 is modified with functional molecules, and astrocytes A are seeded and adhered thereon. Also, the lower surface of the bottom membrane of the cell culture section 38 of the lower substrate 34 is modified with functional molecules, and neurons B are seeded and adhered thereon.

[0074] Similarly, pericytes D are attached to the lower surface of the bottom membrane of the cell culture section 38 of the upper substrate 36, and vascular endothelial cells C are attached to the upper surface.

[0075] 6, the upper substrate 36 is placed on top of the lower substrate 34. At this time, the convex portions 44 of the lower substrate 34 are fitted into the concave portions 54 of the upper substrate 36, and the top surfaces of the convex portions 44 of the lower substrate 34 are brought into contact with the bottom surfaces of the concave portions 54 of the upper substrate 36, thereby achieving horizontal and vertical alignment.

[0076] As described above, the two-layer structure of nervous system tissue in the brain can be reconstructed by co-culturing neurons B and astrocytes A on both sides of the bottom membrane of the cell culture section 38 of the lower substrate 34. Furthermore, the vascular system in the brain can be reconstructed by co-culturing vascular endothelial cells C and pericytes D on both sides of the bottom membrane of the cell culture section 48 of the upper substrate 36. Furthermore, by placing the cell culture membranes of the lower substrate 34 and the upper substrate 36 in close proximity, communication between these cells is promoted. By constructing such a blood-brain barrier model, it becomes possible to analyze the transfer of drugs from the blood to brain tissue.

[0077] In this way, by using the cell culture device of the second invention, cells can be pre-cultured on both sides of the cell culture membranes of the upper and lower substrates, and then the upper substrate can be placed on the lower substrate, making it easy to culture cells in multiple layers. [Example]

[0078] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0079] [Preparation of the lower substrate] A silicon substrate (diameter: approximately 50 mm, thickness: approximately 300 μm) having a (100) crystal orientation on one side and a mirror finish was used to prepare a lower substrate by the following procedure.

[0080] (1) A silicon nitride (SiN) film with a thickness of 1.2 μm was formed on both sides of a silicon substrate by plasma CVD. (2) A positive resist OFPR-800LB 23cp (manufactured by Tokyo Ohka Kogyo Co., Ltd.) was applied to the non-mirror side of the silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask with a 16 x 16 rectangular pattern (each side 690 μm) for etching windows. (3) The silicon substrate was immersed in a developer NMD-3 (2.38% aqueous solution of tetramethylammonium hydroxide; manufactured by Tokyo Ohka Kogyo Co., Ltd.) to develop the resist and pattern an etching window.

[0081] (4) Using the remaining resist layer as a mask, the exposed portions of the SiN film were etched using CF4 gas plasma, and then the resist was removed using acetone. (5) The silicon substrate was immersed in a 25% aqueous solution of tetramethylammonium hydroxide heated to approximately 80°C, and the silicon was etched to a depth of approximately 200 μm along the silicon crystal plane into an inverted square truncated pyramid shape (inverted pyramid shape). (6) Positive resist OFPR-800LB 23cp was applied to the mirror side of the silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask with 208 microholes (diameter 2 μm) per cell culture area. (7) The silicon substrate was immersed in the developer NMD-3 to develop the resist and pattern micro-holes.

[0082] (8) Using the remaining resist layer as a mask, the exposed portions of the SiN film were etched using CF4 gas plasma, and then the resist was removed using acetone. (9) The silicon substrate was immersed in a 25% tetramethylammonium hydroxide aqueous solution heated to approximately 80°C, and the silicon was etched along the silicon crystal plane through the etching window and micropores to form a free-standing SiN membrane (bottom membrane) and a cell culture area with a peripheral wall (convex portion). The final thickness of the bottom membrane was approximately 1 μm, the final diameter of the micropores was approximately 3.5 μm, and the size of the bottom membrane per cell culture area was 300 μm square. (10) A rectangular holder made of polycarbonate was cut out from a silicon substrate and attached to the outer periphery of the substrate with PDMS.

[0083] [Preparation of upper substrate] Next, an upper substrate was prepared using a silicon substrate (diameter: approximately 50 mm, thickness: approximately 300 μm) having a mirror finish on one side and a (100) crystal orientation on the surface, in the following manner.

[0084] (1) A 1.2 μm thick SiN film was formed on both sides of a silicon substrate by plasma CVD. (2) Positive resist OFPR-800LB 23cp was applied to the non-mirror side of the silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask with a 16 x 16 rectangular pattern (570 μm on each side) for etching windows. (3) The silicon substrate was immersed in the developer NMD-3 to develop the resist and pattern an etching window.

[0085] (4) Using the remaining resist layer as a mask, the exposed portions of the SiN film were etched using CF4 gas plasma, and then the resist was removed using acetone. (5) The silicon substrate was immersed in a 25% aqueous solution of tetramethylammonium hydroxide heated to approximately 80°C, and the silicon was etched to a depth of approximately 180 μm along the silicon crystal plane into an inverted square truncated pyramid shape (inverted pyramid shape). (6) Positive resist OFPR-800LB 23cp was applied to the mirror surface of the silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask with a pattern of one microhole (diameter 11 μm) and a recess. (7) The silicon substrate was immersed in the developer NMD-3 to develop the resist, and micro-holes and recesses were patterned.

[0086] (8) Using the remaining resist layer as a mask, the exposed portions of the SiN film were etched using CF4 gas plasma, and then the resist was removed using acetone. (9) The silicon substrate was immersed in a 25% tetramethylammonium hydroxide solution heated to approximately 80°C and etched along the silicon crystal plane until the recess depth reached approximately 150 μm. During this process, etching of the silicon also progressed through the etching windows and micropores, forming a freestanding SiN membrane. Additionally, the addition of 0.01% surfactant Triton X-100 to the etching solution prevented over-etching of the corners of the recesses. The final thickness of the bottom membrane was approximately 1 μm, the final diameter of the micropores was approximately 13 μm, and the size of the bottom membrane per cell-holding section was 150 μm square. (10) A rectangular frame made of PDMS (polydimethylsiloxane) was attached to the outer periphery of the top surface of a silicon substrate. The attachment was achieved by irradiating the SiN film surface on the upper substrate and the PDMS surface with vacuum ultraviolet light to form OH groups on both surfaces, and then bringing the two surfaces into contact with each other to allow a dehydration condensation reaction.

[0087] An actual photograph of the fabricated cell culture device is shown in Figure 7. It was confirmed that the fabricated cell culture device allows easy alignment of the cell culture section of the lower substrate and the cell storage section of the upper substrate by fitting the lattice-shaped convex section of the lower substrate (Figure 7(b)) with the lattice-shaped concave section of the upper substrate (Figure 7(a)). The distance between the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell storage section of the upper substrate was approximately 150 μm.

[0088] [Cell seeding test] Using the cell culture device produced in the above example, the number of cells seeded into the cell culture area of ​​the lower substrate through the micropores formed in the SiN film of the upper substrate was confirmed.

[0089] (Preparation of cell culture device and cell suspension) The SiN film surface was modified with PDL (Poly-D-lysine) by immersing the lower substrate overnight at room temperature in a PDL (Poly-D-lysine) aqueous solution prepared with sterile water at a concentration of 0.1 mg / ml. The surface density of PDL was 0.5 μg / cm2 in PBS (phosphate buffered saline). 2 iMatrix-511 (Matrixome, Inc.), prepared to a concentration of 1000 μg / cm², was deposited on the surface of the lower substrate and placed in an incubator at 37°C with 5% CO₂ for 1 hour to modify the PDL on the SiN membrane surface with laminin-511. PDMS strips (approximately 1 mm thick) were placed on the bottom of a 35 mm dish, and the lower substrate was then placed on top of them. Neurobasal Plus Medium (Life Technologies Corporation) containing 2% B-27 Plus Supplement (Life Technologies Corporation) was then poured into the dish until the lower substrate was completely immersed. The upper substrate was then placed on top of the lower substrate. The lattice-shaped convex portions of the lower substrate were mated with the lattice-shaped concave portions of the upper substrate, and alignment was achieved by abutting the top surfaces of the convex portions of the lower substrate against the bottom surfaces of the concave portions of the upper substrate. The above procedure was repeated to prepare three similar cell culture devices.

[0090] Hippocampal neurons collected from mice were suspended in culture medium and seeded at a density of 1800 cells / cm. 2 , 9000cells / cm 2 , 18000cells / cm 2 Three different concentrations of cell suspensions were prepared so that the following were obtained:

[0091] (Cell seeding test) (1) Three different cell suspensions were introduced into the PDMS frame of the upper substrate of each of the three devices, and neurons were seeded. (2) The device was left in an incubator at 37°C with a CO2 concentration of 5% for 2 hours, and the neurons that had passed through the micropores in the upper substrate and reached the lower substrate were allowed to adhere to the surface of the SiN membrane of the lower substrate. (3) The upper substrate was removed, and the lower substrate was washed with PBS. (4) The lower substrate was immersed in 4% paraformaldehyde-phosphate buffer for 15 minutes to fix the cells, and then washed with PBS. (5) The lower substrate was permeabilized by immersing it in a 0.1% Triton X-100 / PBS solution for 15 minutes, and then washed with PBS. (6) The lower substrate was immersed in a DAPI solution diluted 500 times with PBS at room temperature for 30 minutes to fluorescently stain the cell nuclei of neurons attached to the SiN membrane surface of the lower substrate, and then washed with PBS. (7) The number of neurons in each well (cell culture area) was obtained by counting the DAPI-stained cell nuclei using an inverted fluorescence microscope.

[0092] The results of the cell seeding test are shown in FIG. As shown in Figure 8, the number of wells (cell culture sections) to which single neurons adhered was approximately 20% of the total. This indicates that the effect of using the cell culture device of the present invention is significant, as the number was only a few percent with conventional methods. It was also found that the number of wells from which single neurons were obtained does not depend on the neuron seeding density. Normally, when cells are seeded into a culture vessel, the meniscus of the solution at the outer periphery of the vessel has a significant effect, resulting in different cell densities between the center and the periphery of the vessel. This effect becomes particularly significant when the culture vessel is small. However, it was found that the cell culture method using the cell culture device of the present invention allows cells to be seeded without being affected by the meniscus of the solution. [Industrial Applicability]

[0093] The cell culture device of the present invention can be used for culturing cells and is therefore industrially useful. [Explanation of symbols]

[0094] 1 Cell culture device (first invention) 2 Cell culture device (second invention) 10 Lower board 12 Upper board 14 Cell culture department 16 Lower partition member 18 Micropore 20 Convex part 22 PC holder 24 Cell storage section 26 Micropore 28 Upper partition member 30 recess 32 PDMS frames 34 Lower board 36 Upper board 38 Cell Culture Department 40 Microhole 42 Lower partition member 44 Convex part 46 PC holder 48 Cell Culture Department 50 micropores 52 Upper partition member 54 Recess 56 PDMS frames A. Astrocyte B neurons C vascular endothelial cells D. Pericyte

Claims

1. A lower substrate having a cell culture portion having a bottom membrane; An upper substrate is provided above the lower substrate and has a cell storage section having a bottom membrane with one micro through hole formed therein; A cell culture device characterized in that, when the upper substrate is disposed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are disposed vertically with a predetermined distance therebetween.

2. 2. The cell culture device according to claim 1, wherein a plurality of minute through holes are formed in the bottom film of the lower substrate.

3. 3. The cell culture device according to claim 2, wherein the bottom membrane of the upper substrate and / or the lower substrate is made of a transparent material.

4. The bottom film of the upper substrate and / or the lower substrate may be made of silicon nitride (SiN), silicon oxide (SiO ２ 4. The cell culture device according to claim 3, characterized in that it is made of at least one inorganic material selected from the group consisting of silicon oxynitride (SiON) and silicon oxynitride (SiON).

5. The lower substrate has a convex portion or a concave portion, and the upper substrate has a concave portion or a convex portion, The cell culture device of claim 2, characterized in that the convex or concave portion of the lower substrate engages with the concave or convex portion of the upper substrate, thereby aligning the bottom membrane of the lower substrate with the bottom membrane of the upper substrate.

6. The cell culture device according to claim 2, characterized in that the lower substrate has a plurality of cell culture sections arranged in a lattice pattern, and the upper substrate has a plurality of cell storage sections arranged in a lattice pattern.

7. The cell culture device according to claim 6, characterized in that the upper part of a lower partition member that separates the plurality of lattice-shaped cell culture sections of the lower substrate constitutes a convex portion, and a concave portion corresponding to the upper convex portion of the lower partition member is formed in the lower part of the upper partition member that separates the plurality of cell storage sections of the upper substrate corresponding to the lower partition member.

8. A cell culture method for culturing cells using the cell culture device according to claim 2, comprising: A cell culture method comprising the steps of seeding cells onto a bottom membrane of a cell storage section of the upper substrate, dropping the cells onto the bottom membrane of a cell culture section of the lower substrate through a single micro through-hole formed in the bottom membrane of the cell storage section of the upper substrate, and culturing the cells in the cell culture section.

9. A lower substrate having a cell culture section having a bottom membrane with a plurality of micro through holes formed therein; an upper substrate provided above the lower substrate and including a cell storage section having a bottom membrane with a plurality of micro through-holes formed therein; A cell culture method for culturing cells using a cell culture device in which, when the upper substrate is disposed on the lower substrate, a bottom membrane of the lower substrate and a bottom membrane of the upper substrate are vertically arranged at a predetermined interval, comprising: A cell culture method comprising culturing cells on one or both sides of the cell culture membrane of the lower substrate and culturing cells on one or both sides of the cell culture membrane of the upper substrate.

10. 3. The cell culture device according to claim 2, wherein the diameter of the minute through-holes in the bottom film of the upper substrate is 3 to 50 μm.

11. The cell culture device according to claim 2, characterized in that, when the upper substrate is disposed on the lower substrate, the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell storage section of the upper substrate are arranged vertically with a gap of 5 to 500 μm between them.

12. The cell culture device according to claim 10, characterized in that, when the upper substrate is disposed on the lower substrate, the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell storage section of the upper substrate are arranged vertically with a gap of 5 to 500 μm between them.

13. A cell culture method for culturing cells using the cell culture device according to any one of claims 10 to 12, comprising: A cell culture method comprising the steps of seeding cells onto a bottom membrane of a cell storage section of the upper substrate, dropping the cells onto the bottom membrane of a cell culture section of the lower substrate through a single micro through-hole formed in the bottom membrane of the cell storage section of the upper substrate, and culturing the cells in the cell culture section.

14. 10. The cell culture method according to claim 9, wherein cells are cultured on an upper surface or both surfaces of the cell culture membrane of the lower substrate, and cells are cultured on one surface or both surfaces of the cell culture membrane of the upper substrate.

15. A lower substrate having a cell culture section having a bottom membrane with a plurality of micro through holes formed therein; an upper substrate provided above the lower substrate and including a cell storage section having a bottom membrane with a plurality of micro through-holes formed therein; A cell culture method for culturing cells using a cell culture device in which, when the upper substrate is disposed on the lower substrate, a bottom film of the lower substrate and a bottom film of the upper substrate are vertically spaced apart from each other by a distance of 5 to 500 μm, A cell culture method comprising culturing cells on one or both sides of the cell culture membrane of the lower substrate and culturing cells on one or both sides of the cell culture membrane of the upper substrate.

16. 16. The cell culture method according to claim 15, wherein cells are cultured on an upper surface or both surfaces of the cell culture membrane of the lower substrate, and cells are cultured on one surface or both surfaces of the cell culture membrane of the upper substrate.

Images