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 Art

[0002] Conventionally, in the fields of medicine and drug discovery, in order to analyze the structures and functions of various cells, tissues, and organs in vivo, cells are cultured and analyzed in a cell culture device. As an analysis method using such a cell culture device, for example, a method of forming a large number of minute cell culture parts in an array by a microfabrication technology applying semiconductor processing technology, culturing a single cell in each cell culture part, and analyzing the functions at the single cell level with high throughput, a method of analyzing cell - cell communication by co - culturing multiple types of cells in minute cell culture parts formed by microfabrication technology, and further a method of analyzing pharmacokinetics etc. using tissues or organs reconstructed in minute cell culture parts are known.

[0003] As a cell culture device used for such an analysis method, for example, a cell culture device has been proposed in which the bottom surface of each well of a microwell array is composed of a self - standing film made of SiN by semiconductor processing technology, and a large number of minute through - holes with a diameter of about 3 μm are formed in the SiN film (see Non - Patent Document 1). By culturing a large number of astrocytes on the back surface of this SiN film and a single neuron on the surface of the SiN film in each well, cell - cell communication through the minute through - holes has been enabled, and the physiological activity of a single neuron has been successfully maintained over a long period.

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

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

[0006] However, to form tissues with three or more layers using such a two-layer microfluidic device separated by a porous membrane, it was necessary to place two or more porous membranes within the microfluidic channel. Furthermore, such devices are fabricated by bonding upper and lower microfluidic channels, manufactured using semiconductor processing technology, with porous membranes made of plastic materials such as polyethylene terephthalate (PET). As the number of layers increases, the accuracy of the alignment during bonding deteriorates. In addition, it is difficult to control the distance between layers; for example, even if cells in the first and second layers can be placed close together separated by the porous membrane, it is difficult to bring cells in the second and third layers close enough to an appropriate distance. [Prior art documents] [Patent Documents]

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

[0008] [Non-Patent Document 1] Yusuke Yoshiaki, 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) [Overview of the Initiative] [Problems that the invention aims to solve]

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

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

[0011] In other words, the present invention is as follows: [1] A lower substrate comprising a cell culture section having a bottom membrane, The system comprises an upper substrate disposed above the lower substrate and having a cell-containing portion having a bottom membrane in which micropores are formed, A cell culture device characterized in that, when the upper substrate is placed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged with a predetermined vertical distance between them.

[0012] [2] The cell culture device according to [1] above, characterized in that a single micropore is formed in the bottom membrane of the upper substrate. [3] The cell culture device according to [2] above, characterized in that 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, characterized in that the bottom membrane of the upper substrate and / or lower substrate is made of a transparent material. [5] The cell culture device according to [4] above, characterized in that 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 protrusion or recess, and the upper substrate has a recess or protrusion, A cell culture device according to any one of the above [2] to [5], characterized in that a protrusion or recess of the lower substrate fits with a recess or protrusion 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 grid, and the upper substrate comprises a plurality of cell containment sections arranged in a grid. [8] The cell culture device according to [7] above, characterized in that the upper part of the lower partition member that separates a plurality of grid-like cell culture sections of the lower substrate constitutes a convex portion, and a recess corresponding to the upper convex portion of the lower partition member is formed at the lower part of the upper partition member that separates a plurality of cell housing sections of the upper substrate corresponding to the lower partition member.

[0013] [9] The cell culture device according to [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 containment section of the upper substrate.

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

[11] The cell culture device according to

[10] above, characterized in that 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] A 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 containment section of the upper substrate are arranged with a vertical 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 convex portion or concave portion of the lower substrate and the concave portion or convex portion of the upper substrate are fitted together to align the bottom film of the lower substrate and the bottom film of the upper substrate.

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

[13] above, characterized in that the lower substrate includes a plurality of cell culture portions arranged in a grid pattern, and the upper substrate includes a plurality of cell accommodation portions arranged in a grid pattern.

[15] The cell culture device according to

[14] above, characterized in that the upper part of the lower partition member that partitions the plurality of grid-shaped cell culture portions 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 partitions the plurality of cell accommodation portions of the upper substrate corresponding to the lower partition member.

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

[15] above, characterized in that the lower substrate and the upper substrate are manufactured 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],

[16] above, characterized in that cells are seeded on the bottom film of the cell accommodation portion of the upper substrate, and the cells are dropped onto the bottom film of the cell culture portion of the lower substrate through one micropore formed in the bottom film of the cell accommodation portion of the upper substrate, and the cells are cultured in the cell culture portion.

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

[16] above, characterized in that cells are cultured on one side or both sides of the cell culture film of the lower substrate, and cells are cultured on one side or both sides of the cell culture film of the upper substrate.

Advantages of the Invention

[0015] According to the present invention, it is possible to provide a novel cell culture device and a cell culture method useful for analyzing functions at the single cell level, analyzing cell-cell communication, and the like.

Brief Description of the Drawings

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

Modes for Carrying Out the Invention

[0017] The cell culture device of the present invention includes a lower substrate having a cell culture part with a bottom membrane, and an upper substrate disposed above the lower substrate and having a cell accommodation part with a bottom membrane in which micropores are formed. 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 arranged at a predetermined interval vertically. The cell culture device of the present invention is not limited to one in which the substrates are arranged in two upper and lower layers, and may be one in which three or more layers are stacked.

[0018] The cell culture device of the present invention is useful for analyzing single-cell level functions and intercellular communication, among other applications.

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

[0020] <Cell culture device according to the first invention> The cell culture device according to the first invention will be described below. The cell culture device according to the first 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 containment section with a bottom membrane having a single micropore, characterized in that when the upper substrate is disposed on the lower substrate, the bottom membranes of the lower substrate and the bottom membranes of the upper substrate are arranged with a predetermined vertical distance between them.

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

[0022] The following describes each component of the cell culture device according to the first invention. [Lower circuit board] The lower substrate is a substrate equipped with a cell culture section, and its material can be, for example, a ceramic wafer such as a silicon wafer, glass wafer, or quartz wafer. 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 the area where cells are seeded and cultured, and is, for example, a well (recess) formed in the lower substrate. The shape of the internal space of the cell culture section is not particularly limited, and examples include cylindrical, prismatic, inverted cone-shaped, and inverted pyramidal shape. An inverted pyramidal shape is preferred, and an inverted square pyramidal shape (inverted pyramidal shape) is particularly preferred. One or more cell culture sections can be provided, and usually the number corresponds to the number of cell housing sections in the upper substrate, which will be described later, and they are arranged in positions corresponding to the cell housing sections in the upper substrate. It is preferable that many cell culture sections are arranged in a grid-like (array-like) configuration.

[0024] When the cell culture sections are arranged in a grid pattern, the cell culture sections are partitioned and separated by partition members (lower partition members) that are arranged in a grid pattern and 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 housing sections of the upper substrate by fitting all or part of the upper part of these lower partition members into the recess formed in the lower part of the upper partition member of the cell housing section of the upper substrate, which will be described later.

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

[0026] While it is not necessary for cell culture membranes to have micropores, it is preferable that they have 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 facilitated through the micropores.

[0027] The shape of the micropores can be appropriately set depending on the cell type and culture purpose, for example, circular or polygonal shapes. The diameter of the micropores (or diagonal in the case of polygons) is preferably large enough that cells cannot pass through, 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 micropores in the bottom membrane of the cell culture section depends on the diameter of the micropores, but for example, it is preferably 10 to 70%, more preferably 10 to 50%, and even more preferably 10 to 20%. Within this range, the supply of nutrients and intercellular communication through the micropores can be carried out smoothly, and in the case of transparent materials, high transparency can be maintained.

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

[0029] The material of the cell culture membrane is not particularly limited as long as it serves as a scaffold for the cells, but it is preferable that it be made of a transparent material for ease of cell observation. While the transparent material may be an organic material, an inorganic material is preferred. Examples of transparent inorganic materials include silicon, titanium, zinc, tin, aluminum nitrides, oxides, and oxynitrides. Specifically, examples include silicon nitride (SiN), silicon oxide (SiO2), titanium oxide (TiO2), zinc oxide (ZnO), tin oxide (SnO2), silicon oxynitride (SiON), titanium oxynitride (TiON), indium oxide (In2O3), 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. Furthermore, a laminated film of two or more materials may be formed. For example, a transparent organic material may be coated on the surface of an inorganic material. In this specification, "transparent" means that the light transmittance at wavelengths of 500 nm to 600 nm is 70% or higher, preferably 80% or higher, and more preferably 90% or higher.

[0030] The bottom membrane (cell culture membrane) of the cell culture section is preferably modified on its surface with functional molecules to promote cell adhesion and cell proliferation. Functional molecules are not particularly limited as long as they can be used in cell culture. Examples include extracellular matrix components such as collagen, proteoglycans, heparan sulfate proteoglycans, fibronectin, laminin, enterin, 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 section) The lower substrate preferably includes a support frame (holder) to support it. This allows for more stable placement of the upper substrate when it is mounted on the lower substrate. Furthermore, it can be used as a gripping part, improving operability. The support frame can be made of synthetic resin.

[0032] [Upper circuit board] The upper substrate is disposed above the lower substrate described above and comprises a cell-containing section having a bottom membrane with a single micropore formed therein. The material of the upper substrate can be the same as that of the lower substrate, and may be the same material as the lower substrate or a different material. 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 housing area) The cell containment section is a part that can contain cells, and is, for example, a well (recess) formed in the upper substrate. The shape of the internal space of the cell containment section is not particularly limited, and examples include cylindrical, prismatic, inverted cone-shaped, and inverted pyramidal shape. An inverted pyramidal shape is preferred, and an inverted square pyramidal shape (inverted pyramidal shape) is more preferred. One or more cell containment sections can be provided, and usually the number corresponds to the cell culture section of the lower substrate described above, and they are arranged in positions corresponding to the cell culture section of the lower substrate. It is preferable that many cell containment sections are arranged in a grid (array). If two or more cell containment sections are provided, it is preferable that each cell containment section has the same configuration so that cells fall uniformly.

[0034] When the cell containment sections are arranged in a grid pattern, the cell containment sections are partitioned and divided by partition members (upper partition members) that are arranged in a grid pattern and form the side walls (peripheral walls) of the cell containment sections. As described above, it is preferable to form a recess at the bottom of this upper partition member and fit the protrusion at the top of the lower partition member of the cell culture section of the lower substrate into it to align the two. Normally, when the upper substrate is placed on the lower substrate, a part of the cell containment section of the upper substrate is introduced into the cell culture section of the lower substrate. Therefore, the bottom membrane of the cell containment 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 the size.

[0035] A single micropore, large enough for a cell to pass through, is formed in the bottom membrane of the cell containment section. Preferably, this single micropore is formed in the center of the bottom membrane of the cell containment section. This ensures that when the upper substrate is placed above the lower substrate, the micropore is positioned above the center of the bottom membrane of the cell culture section of the lower substrate, allowing the cells to reliably fall into the cell culture section of the lower substrate. The shape of the micropore can be appropriately set depending on the type of cell, for example, it can be circular or polygonal. The diameter of the micropore (or diagonal in the case of a polygon) can be appropriately set depending on the type of cell, preferably being greater than or equal to the diameter of one cell and less than the sum of the diameters of two cells. For example, a diameter of 3 to 50 μm is preferred, and 8 to 20 μm is more preferred. Specifically, for example, in the case of neurons, since their diameter is about 8 μm, a micropore with a diameter of 10 to 15 μm is preferred. This allows for seeding of only one cell per cell culture section with high probability.

[0036] (Support frame section) The upper substrate preferably includes a support frame (holder) to support it. This allows for more stable placement of the upper substrate when it is mounted on the lower substrate. Furthermore, it can be used as a gripping part, improving operability. The material of the peripheral wall can be a synthetic resin.

[0037] [Alignment configuration of the cell culture section of the lower substrate and the cell containment section of the upper substrate] In the cell culture device according to the first invention, when the upper substrate is placed on the lower substrate, the bottom membranes of the lower substrate and the upper substrate are arranged with a predetermined vertical gap between them, and one micropore of 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 respective bottom membranes coincide. This ensures that cells can be reliably and accurately dropped onto the bottom membrane of the cell culture section of the lower substrate. The gap 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 and upper substrates are aligned is not particularly limited. Examples include fitting together protrusions and indentations formed on the lower and upper substrates, fitting together adjacent periphery protrusions of different diameters (or diagonals in the case of polygons) formed on the outer circumferences of the lower and upper substrates, placing the upper substrate within the periphery frame of the lower substrate, aligning the marks placed on the lower and upper substrates and fixing them with a fastener, and inserting a through rod through the through holes formed on the lower and upper substrates and fixing them.

[0039] Specifically, examples of how the protrusions and indentations formed on the lower and upper substrates are fitted together include a configuration in which the lower substrate has a convex or concave portion, and the upper substrate has a concave or convex portion, and the convex or concave portion of the lower substrate fits together with the concave or convex portion of the upper substrate to align the cell culture portion of the lower substrate with the cell housing portion of the upper substrate. More specifically, examples include a configuration in which the surrounding concave or concave portion surrounding the lower substrate fits together with the surrounding concave or convex portion surrounding the upper substrate, and a configuration in which the upper part of a lower partition member that divides a plurality of lattice-shaped cell culture portions of the lower substrate forms a convex portion, and the lower part of an upper partition member that divides a plurality of cell housing portions of the upper substrate corresponding to the lower partition member has a concave portion that corresponds to the upper convex portion of the lower partition member, and the lattice-shaped convex portion of the lower substrate fits together with the lattice-shaped concave portion of the upper substrate. The convex or concave portion may be provided as one on each of the lower and upper substrates, or as multiple portions.

[0040] One way in which peripheral protrusions formed on the lower and upper substrates are fitted together adjacent to each other is to arrange peripheral protrusions surrounding the lower substrate and peripheral protrusions surrounding the upper substrate, which have a diameter that is slightly larger or smaller than the protrusions on the lower substrate (or the diagonal in the case of a polygon), in an adjacent fitted configuration.

[0041] In this configuration of alignment between the lower and upper substrates, the horizontal and vertical alignment (distance between the cell culture membrane of the upper substrate and the cell containment section of the upper substrate) can be easily and accurately performed without the use of special equipment, thereby improving operability during cell culture.

[0042] <Method for manufacturing a cell culture device according to the first invention> Next, a method for manufacturing the cell culture device according to the first invention will be described. The method for fabricating the lower and upper substrates in the cell culture device according to the first invention is not particularly limited and can be fabricated by various methods, but it is preferable to fabricate them using photolithography, a semiconductor microfabrication technology. For example, a method similar to that for the cell culture sheet described in Japanese Patent Application Publication No. 2014-147342 can be used.

[0043] The following describes an example of a method for manufacturing a cell culture device according to the first invention of this invention. Specifically, the present invention will describe a case in which the cell culture device comprises a lower substrate having a plurality of cell culture sections arranged in a grid, and an upper substrate having a plurality of cell housing sections arranged in a grid, the upper part of the lower partition member that separates the grid-like plurality of cell culture sections of the lower substrate has a convex portion, and the lower part of the upper partition member that separates the plurality of cell housing sections of the upper substrate corresponding to the lower partition member has a recess formed therein that corresponds to the upper convex portion of the lower partition member. Furthermore, the present invention will describe a case in which a silicon substrate with a (100) crystal orientation on its surface and one side being a mirror surface is used as the lower substrate and upper substrate.

[0044] (Fabrication of the lower circuit board) The lower substrate fabrication process includes a cell culture section formation process and a bottom membrane micropore formation process. The cell culture section formation process includes, for example, the steps of: forming a transparent silicon nitride film (inorganic material film) 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, and irradiating the surface of the resist layer with ultraviolet light through a photomask on which multiple etching window patterns are drawn; immersing the silicon substrate in a developer solution to develop the resist layer and pattern the etching windows; using the remaining resist layer as a mask (protective film), etching the exposed silicon nitride film with a plasma-generated reactive gas, and then removing the resist layer with an organic solvent; and immersing the silicon substrate in an etching solution to etch the silicon in a truncated square pyramidal shape (inverted pyramidal shape) along the crystal plane of the silicon, thereby forming multiple cell culture sections in a grid pattern. Simultaneously with the formation of the cell culture sections, lattice-like protrusions (peripheral walls of the cell culture sections) are formed.

[0045] Furthermore, the bottom membrane micropore formation process includes the steps of: applying a photoresist (photosensitive resin) to the silicon nitride film on the other side (mirror surface) of the silicon substrate to form a resist layer; irradiating with ultraviolet light through a glass mask on which multiple micropores are drawn for each cell culture section; immersing the silicon substrate in a developer solution to develop the resist layer and pattern the micropores; and using the remaining resist layer as a mask (protective film), etching the exposed silicon nitride film with a plasma-generated reactive gas to form micropores, and then removing the resist layer.

[0046] Furthermore, it is preferable to have a final finishing step in which the silicon substrate is immersed in an etching solution and the remaining silicon is etched along the silicon crystal plane through the etching window and micropores.

[0047] (Fabrication of the upper circuit board) The upper substrate fabrication process includes a cell containment section formation step and a bottom micropore and recess formation step.

[0048] The cell containment formation process includes, for example, the steps of forming a transparent silicon nitride film (inorganic material film) on both sides of a silicon substrate using a chemical vapor deposition method such as plasma CVD; forming a resist layer by applying a photoresist (photosensitive resin) to the silicon nitride film on one side (non-mirror surface) of the silicon substrate, and irradiating the surface of the resist layer with ultraviolet light through a photomask on which a rectangular pattern for multiple etching windows is drawn; immersing the silicon substrate in a developer solution to develop the resist layer and pattern the etching windows; using the remaining resist layer as a mask (protective film), etching the exposed silicon nitride film with a plasma-generated reactive gas, and then removing the resist layer with an organic solvent; and immersing the silicon substrate in an etching solution to etch the silicon in a truncated square pyramidal shape (inverted pyramidal shape) along the silicon crystal plane from the etching windows, thereby forming a grid of multiple cell containment parts. Similar to the lower substrate, the grid-like protrusions (peripheral walls of the cell containment parts) are formed simultaneously with the formation of the cell containment parts.

[0049] Furthermore, the bottom film micropore and recess formation process includes the steps of: applying a photoresist (photosensitive resin) to the silicon nitride film on the other side (mirror surface) of the silicon substrate to form a resist layer, irradiating with ultraviolet light through a photomask on which a micropore and recess pattern of one per cell containment area is drawn; immersing the silicon substrate in a developer solution to develop the resist layer and pattern the micropores and recesses; using the remaining resist layer as a mask (protective film), etching the exposed silicon nitride film with a plasma-generated reactive gas to form the areas where 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 is added to etch the silicon along the silicon crystal plane to form the recesses.

[0050] Furthermore, it is preferable to perform a finishing process that involves etching the remaining silicon from the cell containment section formation process at the same time as the process of forming the recess.

[0051] <Cell culture method according to the first invention> Next, a cell culture method using the cell culture device according to the first invention described above will be explained. The cell culture method according to the first invention is a cell culture method that uses the cell culture device according to the first invention, characterized in that cells are seeded on the bottom membrane of the cell containment section of the upper substrate, the cells fall onto the bottom membrane of the cell culture section of the lower substrate through a single micropore formed in the bottom membrane of the cell containment section of the upper substrate, and the cells are cultured in the cell culture section.

[0052] According to the cell culture method of the first invention, cells are dropped and seeded into the cell culture section of the lower substrate through a single micropore formed in the bottom membrane of the cell containment section of the upper substrate. This method allows for the introduction of one cell per well with a higher probability than the conventional method of directly seeding cells into a microwell array using a micropipette. Furthermore, while adherent cells such as neurons require a large culture area, this method allows for the introduction of one cell per well with a high probability even into such large cell culture sections.

[0053] Hereinafter, embodiments of a 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, Figure 1 is a schematic diagram illustrating a cell culture device according to one embodiment of the first invention. Figure 2 is a schematic cross-sectional view of the lower and upper substrates of the cell culture device of Figure 1. Figure 3 is an explanatory diagram of a cell culture method using the cell culture device of Figure 1.

[0055] As shown in Figure 1, a cell culture device 1 according to one embodiment of the first invention comprises 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 arranged in a 4x4 grid and comprises 16 cell culture sections 14, each having a bottom membrane measuring 300 μm square (X). The bottom membrane of the cell culture section 14 is a silicon nitride cell culture membrane having approximately 200 micropores 18 with a diameter of approximately 3 μm. The upper part of the lower partition member 16 that divides the grid-like cell culture sections 14 of the lower substrate 10 constitutes a convex portion 20. The lower substrate 10 is equipped 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 arranged in a 4x4 grid and comprises 16 cell containment sections 24 having a bottom membrane of 150 μm square (Y). The bottom membrane of the cell containment section 24 is a silicon nitride film with a single micropore 26 of 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 at the lower part of the upper partition member 28 that partitions the cell containment 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 containment section 24 of the upper substrate 12 is approximately 150 μm, and horizontal and vertical alignment is achieved by fitting the upper protrusion 20 of the lower substrate 10 with the recess 30 of the upper substrate 12. The upper substrate 12 is equipped with a PDMS (polydimethylsiloxane) frame 32 as a support frame that supports the upper substrate 12.

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

[0059] Next, the upper substrate 12 is placed on top of the lower substrate 10 (Figure 3(b)). At this time, the protrusion 20 of the lower substrate 10 and the recess 30 of the upper substrate 12 are fitted together, and the top surface of the protrusion 20 of the lower substrate 10 is brought into contact with the bottom surface of the recess 30 of the upper substrate 12, thereby aligning the substrate horizontally and vertically.

[0060] Next, a neuronal cell suspension is introduced into the cell containment section 24 of the upper substrate 12 to seed neurons B (Figure 3(c)). This allows for the introduction of one neuron B into the cell culture section 14 of the lower substrate 10 with high probability through the micropores 26 of the bottom membrane of the cell containment section 24.

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

[0062] As described above, by using the cell culture device of the first invention, cells can be dropped through a single micropore in the cell containment section of the upper substrate, thereby enabling high-probability seeding of single cells in the cell culture section of the lower substrate. Furthermore, co-culture of a single neuron B and an astrocyte A is performed with the bottom membrane (cell culture membrane) of the cell culture section 14 of the lower substrate 10 in between, and intercellular communication between the two cells is realized through the micropore 18. This makes long-term culture of single neurons possible, enabling analysis and drug efficacy evaluation at the single-neuron level.

[0063] <Cell culture device according to the second invention> Next, I will describe the cell culture device according to the second invention. The cell culture device according to the second invention comprises a lower substrate having a cell culture section with a bottom membrane (cell culture membrane) having a plurality of micropores, and an upper substrate disposed above the lower substrate and having a cell containment section with a bottom membrane (cell culture membrane) having a plurality of micropores, characterized in that when the upper substrate is disposed on the lower substrate, the bottom membranes of the lower substrate and the upper substrate are arranged with a predetermined vertical distance between them.

[0064] In the cell culture device according to the second invention, since the cell culture membrane of the lower substrate and the cell culture membrane of the upper substrate are arranged with a predetermined gap between them vertically, cells can be pre-cultured on one or both sides of the cell culture membranes of the upper and lower substrates, and by stacking these substrates, good intercellular communication can be achieved. Furthermore, it becomes possible to easily stack three or more substrates for 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 multiple micropores are 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, in which the bottom membrane is the bottom membrane of the lower substrate of the cell culture device according to the first invention in which multiple micropores are formed. Furthermore, the manufacturing of the cell culture device according to the second invention is substantially the same as the manufacturing method of the cell culture device according to the first invention, and any changes in the manufacturing method based on changes in the configuration of the device can be applied by appropriately adapting 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 described above will be explained. The cell culture method according to the second invention is a cell culture method that uses the cell culture device of the second invention described above, 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, by using a cell culture device in which cells are pre-cultured on one or both sides of the cell culture membranes of the upper and lower substrates, and the upper substrate is placed on the lower substrate, it becomes possible to easily perform cell culture in three or four layers.

[0068] The following describes in detail an embodiment of a cell culture device according to one embodiment of the second invention with reference to the drawings, but the present invention is not limited to these embodiments.

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

[0070] As shown in Figure 4, the cell culture device 2 according to one embodiment of the second invention comprises 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 arranged in a 4x4 grid and comprises 16 cell culture sections 38 having a bottom membrane of 300 μm square (X). The bottom membrane of the cell culture section 38 is a silicon nitride cell culture membrane having approximately 200 micropores 40 with a diameter of approximately 3 μm. The upper part of the lower partition member 42 that divides the grid-like cell culture sections 38 of the lower substrate 34 constitutes a convex portion 44. The lower substrate 34 is equipped with a PC (polycarbonate) holder 46 as a support frame portion that supports the lower substrate 34.

[0072] As shown in Figures 5 and 6, the upper substrate 36 is arranged in a 4x4 grid and comprises 16 cell culture sections (cell containment sections) 48, each having a bottom membrane of 150 μm square (Y). The cell culture section 48 is composed of a silicon nitride cell culture membrane having approximately 200 micropores 50 with a diameter of approximately 3 μm. A recess 54 corresponding to the upper protrusion 44 of the lower partition member 42 is formed at the bottom of the upper partition member 52 that separates the 16 cell culture sections 48 of the upper substrate 36. 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, and horizontal and vertical alignment is achieved by fitting the upper protrusion 44 of the lower substrate 34 with the recess 54 of the upper substrate 36. The upper substrate 36 is equipped with a PDMS (polydimethylsiloxane) frame 56 as a support frame that supports the upper substrate 36.

[0073] Next, we will describe an example of a cell culture method using the cell culture device 2 described above. First, a functional molecule is applied to the upper surface of the bottom membrane of the cell culture section 38 of the lower substrate 34, and astrocytes A are seeded and attached thereto. Additionally, a functional molecule is applied to the lower surface of the bottom membrane of the cell culture section 38 of the lower substrate 34, and neurons B are seeded and attached thereto.

[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] Next, as shown in Figure 6, the upper substrate 36 is placed on top of the lower substrate 34. At this time, the protrusions 44 of the lower substrate 34 and the recesses 54 of the upper substrate 36 are fitted together, and the top surface of the protrusions 44 of the lower substrate 34 is brought into contact with the bottom surface of the recesses 54 of the upper substrate 36, thereby aligning the substrate horizontally and vertically.

[0076] As described above, 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, the two-layer structure of the nervous system tissue in the brain can be reconstructed. Furthermore, 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, the vascular system in the brain can be reconstructed. In addition, by placing the cell culture membranes of the lower substrate 34 and the upper substrate 36 in extremely 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] Thus, by using the cell culture device of the second invention, it becomes possible to easily perform multilayer cell culture by pre-cultivating cells on both sides of the cell culture membranes of the upper and lower substrates, and then placing the upper substrate on the lower substrate. [Examples]

[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] [Fabrication of the lower substrate] A silicon substrate (approximately 50 mm in diameter and 300 μm thick) with a (100) crystal orientation on its surface and one side being mirror-finished was used to fabricate the lower substrate using the following procedure.

[0080] (1) A silicon nitride film (SiN film) with a thickness of 1.2 μm was formed on both sides of the silicon substrate by plasma CVD. (2) A positive-type 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 on which a rectangular pattern (690 μm per side) for etching windows was drawn. (3) The silicon substrate was immersed in developer NMD-3 (2.38% aqueous solution of tetramethylammonium hydroxide; manufactured by Tokyo Ohka Kogyo Co., Ltd.) to develop the resist and pattern the etching window.

[0081] (4) Using the remaining resist layer as a mask, the exposed SiN film was etched using a CF4 gas plasma, and then the resist was removed using acetone. (5) A 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 in an inverted truncated square pyramidal shape (inverted pyramidal shape) along the crystal plane of the silicon. (6) A positive resist OFPR-800LB 23cp was applied to the mirror side of a silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask in which 208 micropores (2 μm in diameter) were drawn for each cell culture section. (7) The silicon substrate was immersed in developer solution NMD-3 to develop the resist and pattern the micropores.

[0082] (8) Using the remaining resist layer as a mask, the exposed SiN film was etched using a CF4 gas plasma, and then the resist was removed using acetone. (9) A silicon substrate was immersed in a 25% aqueous solution of tetramethylammonium hydroxide heated to approximately 80°C, and the silicon was etched along the silicon crystal plane through etching windows and micropores to form a self-supporting membrane (bottom membrane) made of SiN, as well as a cell culture section with a peripheral wall (protrusions). 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 section was 300 μm square. (10) A rectangular holder made of polycarbonate, which was cut from a silicon substrate and manufactured separately, was bonded to the outer edge of the substrate with PDMS.

[0083] [Fabrication of the upper circuit board] Next, using a silicon substrate (approximately 50 mm in diameter and 300 μm thick) with a (100) crystal orientation on its surface and one side being mirror-finished, the upper substrate was fabricated using the following procedure.

[0084] (1) A 1.2 μm thick SiN film was formed on both sides of the silicon substrate by plasma CVD. (2) A 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 on which a rectangular pattern (570 μm per side) for etching windows was drawn. (3) The silicon substrate was immersed in developer solution NMD-3 to develop the resist and pattern the etching window.

[0085] (4) Using the remaining resist layer as a mask, the exposed SiN film was etched using a 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 in an inverted truncated square pyramidal shape (inverted pyramidal shape) along the crystal plane of the silicon. (6) A positive-type resist OFPR-800LB 23cp was applied to the mirror side of a silicon substrate, and ultraviolet light was irradiated onto the resist surface through a glass mask on which a pattern of one micro-pore (11 μm in diameter) and recesses was drawn. (7) The silicon substrate was immersed in developer solution NMD-3 to develop the resist and pattern the micropores and recesses.

[0086] (8) Using the remaining resist layer as a mask, the exposed SiN film was etched using a CF4 gas plasma, and then the resist was removed using acetone. (9) A silicon substrate was immersed in a 25% aqueous solution of tetramethylammonium hydroxide heated to approximately 80°C, and etched along the silicon crystal plane until the depth of the depressions reached approximately 150 μm. During this process, etching of silicon also progressed through the etching windows and micropores, forming a self-supporting film made of SiN. In addition, over-etching of the corners constituting the depressions was suppressed by adding a 0.01% concentration of the surfactant Triton X-100 to the etching solution beforehand. 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 of one cell containment section was 150 μm square. (10) A rectangular frame made of PDMS (polydimethylsiloxane) was cut from a silicon substrate and bonded to the outer periphery of the top surface. Bonding was performed by irradiating the SiN film surface on the top substrate surface and the PDMS surface with vacuum ultraviolet light to form OH groups on both surfaces, and then bringing the two surfaces into contact and performing a dehydration condensation reaction.

[0087] Figure 7 shows an actual photograph of the fabricated cell culture device. It was confirmed that the fabricated cell culture device allows for easy alignment of the cell culture section of the lower substrate and the cell containment section of the upper substrate by fitting together the grid-like protrusions of the lower substrate (Figure 7(b)) with the grid-like recesses 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 containment section of the upper substrate was approximately 150 μm.

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

[0089] (Preparation of cell culture devices and cell suspensions) The SiN film surface was modified with PDL (Poly-D-lysine) by immersing the substrate overnight at room temperature in an aqueous PDL solution prepared with sterile water to a concentration of 0.1 mg / ml. A surface density of 0.5 μg / cm³ was obtained using PBS (phosphate buffer saline). 2 iMatrix-511 (manufactured by Matrixome Co., Ltd.), prepared to achieve the desired result, was stored on the surface of the lower substrate and left to stand for 1 hour in an incubator at 37°C and a CO2 concentration of 5% to modify the PDL on the SiN membrane surface with laminin-511. A strip-shaped PDMS spacer (approximately 1 mm thick) was placed on the bottom of a 35 mm dish, and the lower substrate was placed on top of it. Neurobasal Plus Medium (manufactured by Life Technologies Corporation), containing B-27 Plus Supplement (manufactured by Life Technologies Corporation) at a concentration of 2%, was introduced until the lower substrate was completely submerged. The upper substrate was then placed on top of the lower substrate. At this time, the grid-like protrusions of the lower substrate and the grid-like recesses of the upper substrate were fitted together, and alignment was performed by bringing the top surface of the protrusions of the lower substrate into contact with the bottom surface of the recesses 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 the seeding density was set to 1800 cells / cm³. 2 9000 cells / cm 2 , 18000 cells / cm 2 Three different concentrations of cell suspensions were prepared to achieve the following results.

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

[0092] The results of the cell seeding experiment are shown in Figure 8. As shown in Figure 8, the number of wells (cell culture sections) to which single neurons were attached accounted for approximately 20% of the total. This is significantly higher than the several percent achieved with conventional methods, demonstrating the effectiveness of the cell culture device of the present invention. Furthermore, it was found that the number of wells to which single neurons were obtained did not depend on the neuron seeding density. Normally, when seeding cells in a culture vessel, the meniscus of the solution at the outer edge of the vessel has a significant impact, resulting in different cell densities in the center and outer edges of the vessel. This effect is particularly pronounced 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 for cell seeding without being affected by the meniscus of the solution. [Industrial applicability]

[0093] The cell culture device of the present invention is industrially useful because it can be used for cell culture. [Explanation of symbols]

[0094] 1. Cell culture device (first invention) 2. Cell culture device (second invention) 10 Lower circuit board 12 Upper circuit board 14 Cell culture department 16 Lower partition member 18 Micropore 20 Convex part 22 PC holder 24 Cell housing section 26 Micropore 28 Upper partition member 30 recesses 32 PDMS frames 34 Lower circuit board 36 Upper circuit 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 frame Astrocyte B neuron C vascular endothelial cells D Perisite

Claims

1. A lower substrate comprising a cell culture section having a bottom membrane, The system comprises an upper substrate disposed above the lower substrate and having a cell-containing section having a bottom membrane in which one micro-through-hole is formed, A cell culture device characterized in that, when the upper substrate is placed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged with a predetermined vertical distance between them.

2. The cell culture device according to claim 1, characterized in that a plurality of micro-through-holes are formed in the bottom membrane of the lower substrate.

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

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

5. The lower substrate has a protrusion or recess, and the upper substrate has a recess or protrusion, The cell culture device according to claim 2, characterized in that the protrusion or recess of the lower substrate and the recess or protrusion of the upper substrate are fitted together to align the bottom membrane of the lower substrate and the bottom membrane of the upper substrate.

6. The cell culture device according to claim 2, characterized in that the lower substrate comprises a plurality of cell culture sections arranged in a grid, and the upper substrate comprises a plurality of cell containment sections arranged in a grid.

7. The cell culture device according to claim 6, characterized in that the upper part of the lower partition member that separates a plurality of grid-like cell culture sections of the lower substrate constitutes a convex portion, and a recess corresponding to the upper convex portion of the lower partition member is formed at the lower part of the upper partition member that separates a plurality of cell housing sections of the upper substrate corresponding to the lower partition member.

8. A cell culture method comprising culturing cells using the cell culture device described in claim 2, A cell culture method characterized by seeding cells on the bottom membrane of the cell containment section of the upper substrate, dropping the cells onto the bottom membrane of the cell culture section of the lower substrate through a single micro-through-hole formed in the bottom membrane of the cell containment section of the upper substrate, and culturing the cells in the cell culture section.

9. A lower substrate comprising a cell culture section having a bottom membrane in which a plurality of microperforations are formed, The system comprises an upper substrate disposed above the lower substrate and having a cell-containing portion having a bottom membrane in which a plurality of micro-through-holes are formed, A cell culture method for culturing cells using a cell culture device in which, when the upper substrate is placed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged with a predetermined vertical distance between them, A cell culture method characterized by 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. The cell culture device according to claim 2, characterized in that the diameter of the micro-through-pores in the bottom membrane 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 placed on the lower substrate, the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell containment section of the upper substrate are arranged with a vertical 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 placed on the lower substrate, the bottom membrane of the cell culture section of the lower substrate and the bottom membrane of the cell containment section of the upper substrate are arranged with a vertical gap of 5 to 500 μm between them.

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

14. The cell culture method according to claim 9, characterized in that cells are cultured on the upper 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.

15. A lower substrate comprising a cell culture section having a bottom membrane in which a plurality of microperforations are formed, The system comprises an upper substrate disposed above the lower substrate and having a cell-containing portion having a bottom membrane in which a plurality of micro-through-holes are formed, A cell culture method for culturing cells using a cell culture device in which, when the upper substrate is placed on the lower substrate, the bottom membrane of the lower substrate and the bottom membrane of the upper substrate are arranged with a vertical gap of 5 to 500 μm, A cell culture method characterized by 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. The cell culture method according to claim 15, characterized in that cells are cultured on the upper 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.

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