JP2019180354A - Method for producing cell sheet and cell sheet - Google Patents

Abstract

To provide a method for producing a cell sheet containing hepatic cells or intestinal tract cells, the functions of which are more similar to those of in vivo cells.SOLUTION: A method for producing a cell sheet includes culturing hepatic cells or intestinal tract cells, using a cell culture support having a planar mesh structure, wherein the mesh structure has an opening of such a size as to allow passage of at least one cell.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a cell sheet using hepatocytes or intestinal cells, and a cell sheet.

  Attempts have been made to apply to various medical and drug discovery technologies by producing higher order cell structures using cultured cells. Typical examples include various cell sheet applications and medical / drug discovery. The cell sheet is usually an at least single-layer sheet-like cell aggregate formed by mutual bonding between adjacent cells. Cell sheets have begun to be widely used in fields such as regenerative medicine and drug discovery.

  On the other hand, it is not easy to produce a good quality cell sheet. Conventionally, a method in which target cells are seeded in a petri dish or the like and allowed to proliferate is used. However, if the number of cells increases and becomes confluent (very close and dense), cell contact inhibition (contact inhibition) occurs, so passage is necessary, resulting in labor and cost problems. . In some cases, the above problems can have a significant impact on the quality of the cell sheet.

  In the method of culturing using a container such as a petri dish, since the cells adhere to the container, it is necessary to peel the obtained cell sheet from the container. In particular, when cells are strongly bound to the container, it is necessary to add mechanical or chemical operations to recover the cell sheet. Furthermore, quality is important when cell sheets are used for regenerative medicine and drug discovery. However, in the conventional method of producing a cell sheet on the surface of a container such as a petri dish, dead cells cannot be selectively removed, and only a cell sheet in which live cells and dead cells are mixed can be produced. In addition, as described above, cell deterioration due to contact inhibition also occurs, so further improvement was necessary to adapt to regenerative medicine and drug discovery.

  In order to solve the above-described problems, it has been proposed to use a cell culture support having a planar mesh structure, in which the mesh structure openings are larger than the cells to be cultured (Patent Document 1). . In practice, mouse embryonic fibroblasts (MEF), human embryonic lung fibroblasts (TIG120), and mouse embryonic stem cells (ES cells) are cultured and propagated. When cells are seeded in such a state that the cell culture support is suspended in the culture solution, the cells spontaneously extend to the openings of the mesh structure, and the contacted cells adhere and connect to each other to form the openings of the mesh structure. Filled, a single layer cell sheet is formed. Even if the cells proliferate, dead cells and poorly conditioned cells are naturally removed from the cell culture support, so that the culture can be continued for a long period of time without subculture. As a result, a highly safe cell sheet consisting only of living cells in good condition is obtained. Furthermore, it is said that the strength and flexibility of the cell sheet can be increased by adjusting the angle in the incubator.

  In addition, it has been reported that trophoblast cells are induced by culturing iPS cells using a mesh (Non-patent Document 1).

  In drug discovery, the importance of using mature human cells in ADME tests (absorption, distribution, metabolism, excretion tests) and toxicity tests has been recognized. It is still difficult to obtain and culture.

International publication WO2015 / 005394 pamphlet

Okeyo KO et al. Tissue Eng Part C Methods. 2015 Oct; 21 (10): 1105-15. Soldatow, V.Y., et al., In vitro models for liver toxicity testing.Toxicol Res (Camb), 2013. 2 (1): p. 23-39.

  Laboratory animals, normal human hepatocytes and intestinal cells, human established hepatocytes and intestinal intestinal cells are used to evaluate the toxicity and pharmacokinetics of chemical substances such as new drugs. Regarding animal experiments, it has been clarified that there are large species differences in metabolism and excretion of chemical substances, and it is recognized that it is difficult to obtain accurate information on human toxicity, metabolism and excretion from laboratory animals. Has been. In addition, normal human hepatocytes and intestinal cells derived from surgical patients and the like have a large individual difference and cannot be obtained stably and at low cost. Human cell lines have the advantage of easy growth culture and management in vitro (in vitro), but have the disadvantage that their functions are significantly reduced compared to normal cells in vivo.

  As a method for solving these problems, research has been actively conducted to bring the cell line of a human cell line closer to the state of a cell in a living body by three-dimensional culture. There is a spheroid culture method as a general three-dimensional culture method, but as the spheroid (cell mass) becomes larger, it becomes difficult for oxygen and nutrients to reach the inner cell group, and further, it becomes difficult to observe the internal microscope. Therefore, the development of a new three-dimensional culture method is desired (Non-patent Document 2).

  For the purpose of evaluating the absorption of a test compound from the small intestine, a system in which Caco-2 cells are cultured on a polycarbonate membrane or the like having many holes is used. However, in this culture system, Caco-2 cells adhere and spread flatly on a substrate with a hole, and thus can basically be regarded as a two-dimensional culture system. In such a culture system, the expression level of the transporter in Caco-2 cells is not sufficient, and thus the kinetics of the test compound cannot be accurately evaluated in many cases. In addition, since the porosity of the porous membrane generally used for Caco-2 cells is around 15%, transepithelial electrical resistance and compound permeation obtained from the Caco-2 cell sheet on this porous membrane It is considered that the degree information has a different property from those in the living body.

  This invention makes it the subject which should be solved to provide the manufacturing method of the cell sheet containing the hepatocyte or intestinal cell which has a function close | similar to a biological body.

  The present inventors have intensively studied to solve the problem that the maturity of cells is insufficient with many techniques such as spheroids described in Non-Patent Document 2, for example. As a result, hepatocytes or intestinal cells are cultured using a cell culture support having a planar mesh structure, the opening of the mesh structure having a size that allows passage of at least one cell. As a result, it was found that a cell sheet containing hepatocytes or intestinal cells having an unexpectedly high maturity can be produced, and the present invention has been completed. According to the present invention, the following inventions are provided.

<1> A cell culture support having a planar mesh structure, wherein an opening of the mesh structure has a size that allows at least one cell to pass through, so that hepatocytes or intestinal cells are obtained. A method for producing a cell sheet, comprising culturing.
<2> The method for producing a cell sheet according to <1>, comprising culturing the cell culture support in contact with the hepatocytes or intestinal cells.
<3> The method for producing a cell sheet according to <1> or <2>, wherein the cell culture support is placed in a medium and the hepatocytes or intestinal cells are cultured.
<4> The method for producing a cell sheet according to any one of <1 to 3, wherein the hepatocytes or intestinal cells grow in the opening of the cell culture support.
<5> The method for producing a cell sheet according to any one of <1> to <4>, wherein the cell culture support having a planar mesh structure is plate-shaped and has a thickness of 1 μm to 50 μm. .
<6> The cell sheet according to any one of <1> to <5>, wherein the cell culture support having the planar mesh structure is plate-shaped and has a large number of polygonal openings in plan view. Manufacturing method.
<7> The cell culture support having the planar mesh structure according to any one of <1> to <6>, wherein a width of the frame dividing the opening in a plan view is 1 μm or more and 50 μm or less. A method for producing a cell sheet.
<8> In a plan view, the frame is sandwiched between a plurality of substantially parallel straight lines extending in the horizontal direction and two horizontal straight lines and extending in the right diagonal direction, and in the left diagonal direction A straight line extending in a continuous manner forms a broken line structure so as to pass between the two horizontal straight lines, and a triangle is formed by the horizontal straight line, the right diagonal line, and the left diagonal line. Thus, the method for producing a cell sheet according to <7>, wherein a large number of triangular openings are arranged in the support.
<9> The cell according to <8>, wherein the numerous triangular openings formed in the support are substantially equilateral triangles, and the length of one side of the substantially equilateral triangular openings is 30 μm or more and 500 μm or less. Sheet manufacturing method.
<10> The method for producing a cell sheet according to any one of <1> to <9>, wherein the cell culture support having the planar mesh structure is a plate-like porous body made of metal or resin.
<11> Any one of <1> to <10>, wherein the cell culture support is placed in a culture tank, a medium is continuously supplied to and discharged from the culture tank, and a flow is formed in the culture tank. The manufacturing method of the cell sheet of the cell of description.
<12> The method for producing a cell sheet according to <11>, wherein an apparatus having any one of the following structures (a) to (c) is used as an incubator for culturing the hepatocytes or intestinal cells.
(A) It has one reaction plate and a culture tank, and at least one groove leading to the inside of the culture tank is cut in the plate, and a planar mesh structure is formed at the approximate center of the culture tank. The cell culture support can be installed in a form that floats from the bottom of the culture tank, and the culture tank can be filled with a medium so that the support is immersed, and the planar mesh is inserted through the groove of the plate. Cells are cultured on the planar mesh structure of the cell culture support while removing the waste by supplying the medium from the bottom of the structure and discharging the medium from the tube provided at the top of the culture tank. be able to.
(B) It has one reaction plate, and a culture tank is formed at the approximate center of the plate, which is a depression or opening for storing the culture medium. A cell culture support having a structure can be installed, and the reaction plate has two or more grooves extending radially from the culture tank. The grooves serve as flow paths to supply and discharge the medium. By continuously feeding a fresh medium to the culture tank, cells can be cultured on the planar mesh structure of the cell culture support while removing waste products.
(C) It has two or more reaction plates, and at least one of the plates has a culture tank composed of a depression or an opening for storing the culture medium, and a planar shape at the approximate center of the culture tank. A cell culture support having a mesh structure can be installed, and each reaction plate is provided with one or more grooves extending radially from the culture tank. Cells are cultured on the planar mesh structure of the cell culture support while feeding and discharging from the groove of the plate in the same medium, different medium, or a combination of medium and gas, encouraging different changes above and below the cells be able to.
<13> The cell culture support having the planar mesh structure can be arranged at the center of the apparatus, and three or more radial flow paths are formed toward the center support. The method for producing a cell sheet according to <12>, wherein the supply and the discharge are performed through the three or more radial flow paths.
<14> The method for producing a cell sheet according to any one of <1> to <13>, wherein intestinal cells are cultured together with fibroblasts using the cell culture support having the planar mesh structure.
<15> A cell sheet comprising a member having a planar mesh structure having a size that allows at least one cell to pass through the opening, and hepatocytes or intestinal cells.
<16> A cell sheet produced by the method according to any one of <1> to <14>.
<17> The cell sheet according to <15> or <16>, which is stored frozen.

  According to the present invention, by using a support having a planar mesh structure, a cell sheet containing hepatocytes and intestinal cells that are important in drug discovery can be provided.

FIG. 1A is a plan view of a micromesh fixed with a Kapton (registered trademark) tape and a partially enlarged view thereof. FIG. 1B is a perspective view showing a mode in which cells are seeded on a micromesh. FIG. 1C is a perspective view showing an embodiment of an incubator when a micromesh is installed. FIG. 1D is a cross-sectional view showing one embodiment of the incubator when the micromesh is installed. FIG. 2 is a microscopic image showing an aspect of micromesh culture using HepG2 cells in two types of mesh sizes A and B. FIG. 3A is a cross-sectional view taken along the line A ′ showing a block perfusion device for micromesh culture. FIG. 3B is a plan view (partially exploded view) showing a block perfusion device for micromesh culture. FIG. 3C is a cross-sectional view taken along line C ′ showing a block perfusion device for micromesh culture. FIG. 3D is a perspective view showing a micro permeation culture block perfusion device. FIG. 3E is a cross-sectional view taken along line E ′ showing the micromesh culture chip-type perfusion device. FIG. 3F is a plan view showing a chip-type perfusion device for micromesh culture. G in FIG. 3 is a cross-sectional view taken along line G ′ showing a micromesh culture chip-type perfusion device. FIG. 3H is a perspective view showing a chip perfusion device for micromesh culture. FIG. 4 is a graph showing the relative expression level of the liver maturation marker gene in the established hepatocyte HepG2 cell. FIG. 5 is a graph showing the relative expression level of the liver maturation marker gene in the established hepatocyte HepG2 cells cultured using two types of perfusion devices. FIG. 6 is a graph showing the relative expression level of the intestinal maturation marker gene in established intestinal cell Caco-2 cells. FIG. 7 is a microscopic image showing a PDMS micromesh sheet. FIG. 8 is a microscopic image showing an embodiment in which Coco-2 cells are cultured on a PDMS micromesh. FIG. 9A is a cross-sectional view taken along line A ′ of an apparatus for measuring transepithelial electrical resistance (TEER) of a cell sheet formed by micromesh culture. FIG. 9B is a plan view of an apparatus for measuring transepithelial electrical resistance (TEER) of a cell sheet formed by micromesh culture. FIG. 9C is a cross-sectional view taken along line C ′ of an apparatus for measuring transepithelial electrical resistance (TEER) of a cell sheet formed by micromesh culture. FIG. 10 is a graph showing measured values of transepithelial electrical resistance (TEER) in a cell sheet formed by micromesh culture. FIG. 11A is a cross-sectional view taken along line A ′ showing a device for separately perfusing two types of culture solutions above and below a cell sheet formed by micromesh culture. FIG. 11B is a plan view showing a device for separately perfusing two types of culture solutions above and below a cell sheet formed by micromesh culture. FIG. 11C is a transparent perspective view showing a device for separately perfusing two types of culture solutions above and below a cell sheet formed by micromesh culture. FIG. 12 is a microscopic image comparing the differences between spheroid culture and micromesh culture. FIG. 13 is a process explanatory view schematically showing a whole image of a micromesh manufacturing method by a perspective view. FIG. 14 is a process explanatory view schematically showing a part of a process of manufacturing a photomask using photolithography in a cross-sectional view. FIG. 15 is a microscopic image (right figure: enlarged) showing the results of culturing HepG2 cells with micromesh.

The form for implementing this invention is demonstrated below.
In the method for producing a cell sheet of the present invention, a cell culture support having a planar mesh structure, wherein the opening of the mesh structure has a size that allows passage of at least one cell, is used. Then, hepatocytes or intestinal cells are cultured.

  The cell sheet formed by culturing cells using a cell culture support having a planar mesh structure is considered to be excellent in supplying oxygen and nutrients to cells because it is thinner than spheroids. It is estimated that cells with high maturity can be obtained. In addition, it is considered that the layered structure of the in vivo tissue can be reconstructed from the feature of the cell sheet. Furthermore, the cell sheet produced by the micromesh culture has an advantage that it can be easily observed with a microscope from both the upper surface and the lower surface. In addition, since it is a cell sheet with a small cell-substrate adhesion rate, it may be useful for analysis of permeability of the test substance. In connection with this, it is possible to perfuse different fluids separately on the top and bottom of the cell sheet, which is considered suitable for analysis of drug permeability.

(Cell culture support)
Planar mesh structure The cell culture support that can be used in the present invention has a planar mesh structure.
By using a cell culture support having a planar mesh structure, it is possible to provide a culture method with less freezing and thawing damage to cells compared to the spheroid culture method.

  Each opening of the mesh structure has a size that allows cells to be cultured (at least one cell) to pass through. When the cell culture support is placed in a medium (culture solution), cells can be propagated along the shape. The cells spontaneously extend in the openings of the mesh, as compared with the conventional scaffolds in which the cells settle and proliferate on a scaffold having a larger area than the cells. Conventionally, since a scaffold having a smaller opening than cells such as pores (scaffold) is used as a culture support on the bottom surface of a uniform container, the cells settle on the scaffold and proliferate. Form. Therefore, it is difficult to form a sheet only by adhesion between cells. In contrast, when a support having a mesh structure having an opening larger than a cell is used, the cells spontaneously extend toward and adhere to the center of the opening. As a result, it becomes easy to form a single-layer cell sheet so as to cover the opening. In the present specification, “cell extends” or “cell grows” means that the cell deforms, grows or proliferates in a specific direction.

-Opening part In a planar mesh structure, the planar structure in which the opening part of predetermined shape repeated regularly or irregularly is preferable. In the planar mesh structure, it is more preferable to have a large number of polygonal openings in plan view. The opening having the predetermined shape is typically a regular polygon such as a regular triangle, a square, or a regular hexagon, but may be a circle, an ellipse, or another polygon. In addition, in this specification, when referring to a regular triangle, a square, a regular hexagon, and a regular polygon, they are not limited to a regular triangle, a square, a regular hexagon, and a regular polygon in a strict sense. It includes triangles, approximately squares, approximately regular hexagons, and approximately regular polygons.

  The openings may not all have the same shape, and may be a planar structure in which a plurality of openings are set as one set and such sets are regularly or irregularly arranged.

  A portion other than the opening portion of the planar mesh structure is referred to as a frame or a frame portion. The planar mesh structure may be referred to as a micromesh when the opening is a micrometer size.

  The opening of the cell culture support having a planar mesh structure has a size that allows at least one cell to be cultured to pass through. The size that the opening can pass through at least one cell to be cultured means that each size of the opening and the cell can pass through the opening with or without deformation of the cell. To do. The size of the opening may be such that cells can pass without contacting the opening, or the size that allows the cell to pass while deforming in contact with the opening.

  As an example in which the opening has a size that allows at least one cell to be cultured to pass through, the area of the opening of the mesh structure is larger than the maximum area of one cell at the time of seeding. The maximum area of one cell means the area when the cell is cut so that the cross-sectional area is maximized. The maximum area of the cells at the time of seeding means the maximum area when the cells are seeded, that is, before the cells adhere to the support and spread.

In addition, the cell culture support having a planar mesh structure may have a minimum diameter of the opening larger than a maximum diameter when seeding cells to be cultured. The minimum diameter of the opening can be appropriately measured by those skilled in the art. For example, in the case of a regular polygon, the minimum diameter of the straight line connecting two points on the side through the center is referred to. The maximum cell diameter refers to the length of the longest straight line connecting two points around the cell.
When there are two or more types of openings, the minimum diameter of the openings may be larger than the maximum diameter at the time of seeding the cells to be cultured only in any one of the openings. The minimum diameter may be larger than the maximum diameter at the time of seeding cells to be cultured.

  If the minimum diameter of the opening is larger than the maximum diameter of the cell, if the cell culture support is placed horizontally and seeded from above, some cells will settle in the frame and fall down without touching the frame There are also cells. Although only the cells settled in the frame part do not initially fill the opening, the cells spontaneously extend toward the center of the opening, resulting in the formation of a cell sheet.

  In the cell culture support, the minimum diameter of the opening may be 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, or the like of the maximum diameter of cells to be cultured. Also in such a case, the cells seeded in the frame part spontaneously extend to the opening part, and the cells are connected to fill the opening part to form a cell sheet.

  When the opening is a regular polygon, the length of one side is not particularly limited, but can be, for example, about 30 to 500 μm, preferably about 30 to 250 μm. By adjusting the length of one side, the time until the cell sheet is formed, the strength and flexibility of the cell sheet can be controlled. The length of one side may be 50 μm or more, 60 μm or more, 80 μm or more, or 100 μm or more. Moreover, as an upper limit, it is good also as less than 500 micrometers, less than 250 micrometers, less than 200 micrometers, less than 180 micrometers, less than 150 micrometers.

  As described above, the openings of the mesh structure can be triangular, quadrangular, hexagonal, etc., and this shape also controls physical properties such as the time until the cell sheet is formed and the strength and flexibility of the cell sheet. Is possible. As a preferred specific example, a large number of triangular openings formed in a mesh structure are substantially equilateral triangles, and the length of one side of the substantially equilateral triangular openings is 30 μm or more and 250 μm or less.

-Frame The width of the frame portion of the mesh structure of the cell culture support having a planar mesh structure is not particularly limited, and may be, for example, half or less of the maximum cell diameter. As described above, in the conventional scaffold, a structure having a width equal to or larger than the maximum diameter of the cell is used so that the cell settles. However, in the cell culture support having a planar mesh structure, A mesh structure having a frame portion thinner than the size may be used.

  In this specification, the “width of the frame portion” means a width in a direction perpendicular to the length direction of the frame. The width of the frame portion may be one third or less, one fourth or less, one fifth or less, or less than the maximum cell diameter.

  In addition, the width of the frame portion can be set to, for example, 1 μm to 50 μm, and may be 2 μm to 15 μm, 3 μm to 10 μm, 4 μm to 5 μm, and the like, but is not limited thereto. A person skilled in the art can appropriately determine the size according to the cell size. The strength and flexibility of the cell sheet can also be controlled by adjusting the width of the frame portion. Moreover, since the cells adhere to the frame portion when the cells are seeded for the first time, the number of cells to be adhered can be controlled by adjusting the width of the frame portion.

  In this way, by using a mesh structure having a relatively thin frame portion and a relatively large opening portion, cells spontaneously extend into the opening portion, and adjacent cells adhere to each other to form a single-layer cell sheet. Is formed.

  In this specification, the “single-layer cell sheet” refers to a structure in which cells are arranged in at least approximately two dimensions and adjacent cells adhere to each other with almost no gap. Whether or not the cell sheet is a single layer can be confirmed, for example, by staining cell nuclei and arranging the cell nuclei approximately in a plane. The shape may be a shape that can be recognized as a single layer as a whole, and some cells may be layered.

  The cell culture support is preferably plate-shaped, and the thickness thereof may be smaller than the maximum diameter of the cells adhered to the cell culture support. For example, it may be 1 μm to 50 μm, and may be 2 μm to 15 μm, 3 μm to 10 μm, 4 μm to 5 μm, etc., but is not limited thereto. You may determine the thickness of a support body according to the use of the cell sheet obtained.

  In one embodiment of the cell culture support having a planar mesh structure, the frame is sandwiched between a plurality of substantially parallel straight lines extending in the lateral direction and two lateral straight lines in plan view, A straight line that extends diagonally upward to the right from the lower horizontal line to the upper horizontal line (straight line that extends in the diagonally right direction), and diagonally downward to the right from the upper horizontal line to the lower horizontal line following this diagonally right upward line A straight line extending in a diagonal direction (a straight line extending in the diagonally left direction) forms a polygonal line structure continuously zigzag so as to travel between the two horizontal lines, and a horizontal line and a right diagonal line A triangle is formed by a straight line in the diagonally left direction, whereby a large number of triangular openings are arranged in the support body. As an example, when the vertices of a broken line (triangle vertices) continuous in a zigzag between the two horizontal lines are drawn on a virtual line perpendicular to the horizontal line, they coincide with each other on the virtual line, or every other line May match.

  Examples of the structure in plan view as a typical example of the cell culture support having the planar mesh structure of the present embodiment include the micromesh of FIG. 1A and the micromesh of FIG. 2A.

  However, the cell culture support having the planar mesh structure of the present invention is not limited to this. For example, the mesh structure may have shape anisotropy. When the mesh structure is asymmetrical, anisotropy occurs and the cell orientation can be controlled, so that a cell sheet having a desired ordered structure can be obtained. On the contrary, depending on the shape of the mesh structure, cells expand isotropically, and a cell sheet having no orientation can be obtained.

-Material for Cell Culture Support and Manufacturing Method FIG. 13 is a perspective view (bird view) schematically showing a process of obtaining a metal micromesh sheet. The outline of an electroformed mesh manufacturing process is shown. This process is: 1. Apply a photoresist on the substrate. 2. UV exposure using a photomask; 3. Development of photoresist. 4. Electroforming, Photoresist stripping, 6. 6. Strip the electroformed sheet from the substrate. Progress in the form of completion. The photomask is appropriately produced in a desired form by a precision processing manufacturer. The process of FIG. 14 is a more general photomask manufacturing method. 1. Chromium is deposited on a glass substrate. 2. Photoresist application, 3. electron beam drawing or laser drawing; Development, 5. 5. Chrome etching, 6. photoresist removal; Progress in the form of completion. For example, 2.5 inches (6.35 cm), a Cr blank mask, and a positive resist (photosensitive material) attached can be used for the substrate.

As a material for the cell culture support having a planar mesh structure, for example, a photocurable resin, a biocompatible material, a biodegradable material, or the like can be used.
If a photocurable resin is used, a cell culture support can be produced by photolithography.

  Examples of the photocurable resin include acrylate compounds, methacrylate compounds, epoxy compounds, isocyanate compounds, thiol compounds, silicone compounds, and the like. Two or more of these may be used in combination. Specific examples of the photocurable resin include urethane acrylate, polyester acrylate, epoxy acrylate, poly (meth) acrylate methyl, ethoxylated bisphenol A acrylate, aliphatic urethane acrylate, polyester acrylate, polyethylene terephthalate, polystyrene, polycarbonate, acrylic modification. Examples include, but are not limited to, alicyclic epoxides, bifunctional alcohol ether type epoxides, acrylic silicones, and acrylic dimethylsiloxanes.

  A micromesh sheet can be manufactured using a positive resist from which the exposed portion is removed. For example, DNQ (diazonaphthoquinone) novolak resin positive resist, deprotection reaction type such as t-butoxycarbonyl, tetrahydropyran, phenoxyethyl, trimethylsilyl, t-butoxycarbonylmethyl, solutions such as polyphthalaldehyde, polycarbonate, polysilier lutel, etc. A polymerization reaction type is mentioned. Tetramethylammonium hydroxide (TMAH), dimethyl sulfoxide (DMSO), MEK (mel ethyl ketone), GBL (γ-butyrolactone), EL (ethyl lactate), etc. are used as the developer.

  A cell sheet obtained using a biocompatible material or a biodegradable material is suitably used for regenerative medicine and drug discovery, such as transplanting a support body together with a support as it is.

  Examples of biocompatible materials include silicone, polyether block amide (PEBAX), polyurethane, silicone-polyurethane copolymer, ceramics, collagen, hydroxyapatite, nylon, polyethylene terephthalate, Gore-Tex (trademark) and other ultra-high molecular weight polyethylene, Examples include, but are not limited to, polyvinyl chloride and other biological materials. The cell culture support having a planar mesh structure may be formed of a material other than the biocompatible material, and the surface may be treated with the biocompatible material.

  Biodegradable materials include polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and copolymers thereof, PHB-PHV poly (alkanoic acids), polyesters, starch, cellulose, chitosan Examples thereof include natural polymers such as, but are not limited to.

Among the materials described above, polydimethylsiloxane (PDMS) having low autofluorescence is preferred for observation under a fluorescence microscope as the material for the cell culture support having a planar mesh structure.
It is also preferable to produce a cell culture support having a planar mesh structure from a material that can be elastically deformed, such as PDMS. The cell culture support having a planar mesh structure can be devised in various ways, for example, by reinforcing the surroundings to facilitate carrying, and the support added with such a contrivance is also included in the present invention.

  FIG. 14 shows an example in which a desired photomask is manufactured using the glass substrate 101. A vapor deposition layer 102 of a metal such as chromium is attached on the glass substrate 101, and a positive type photoresist is applied thereon. A positive resist is irradiated with a desired pattern of light 105 by a laser direct irradiation device. Only the irradiated portion 103a is softened. There, it develops by apply | coating developers, such as tetramethylammonium hydroxide (TMAH), and the pattern H1 can be obtained. Furthermore, using the remaining resist 103b as a mask, the metal layer can be further etched by, for example, wet etching or dry etching to form the pattern H2. Next, when the remaining resist 103b is also removed with a predetermined chemical solution, a metal layer 102b having a pattern H2 applied on the glass substrate is obtained. This can be used as a photomask. For example, by making the pattern H2 a structure corresponding to a micromesh having continuous regular triangles, a metal film having a continuous micromesh structure having regular triangles as illustrated above can be obtained using this photomask. .

(Cell sheet manufacturing method and cell culture apparatus)
In the method for producing a cell sheet of the present invention, it is preferable to culture in a state where hepatocytes or intestinal cells are brought into contact with the cell culture support.
In the method for producing a cell sheet of the present invention, it is preferable to place a cell culture support in a medium and culture hepatocytes or intestinal cells.
In the method for producing a cell sheet of the present invention, it is preferable that hepatocytes or intestinal cells grow in the opening of the cell culture support.

  One embodiment of the culturing method includes a method comprising a step of seeding cells on a cell culture support and a step of culturing the cells. At this time, in the present embodiment, it is preferable to use a cell culture support having a planar mesh structure and to install the cell culture support in a cell culture apparatus (incubator) prior to the step of seeding cells. The cell culture device and the cell culture support are preferably sterilized as appropriate. A culture solution can be appropriately selected and prepared by a person skilled in the art according to the type of cell. Culture fluid and other materials may be sterilized by autoclaving or filtration.

  The cell culture support may be coated with a cell adhesion promoting material in advance. Cell adhesion promoting materials are used to fix cells on a culture support to facilitate spreading and proliferation. For example, extracellular matrix proteins such as collagen, fibronectin and laminin, positively charged substances such as poly-L-lysine, etc. Is mentioned.

  The cell culture support that has undergone the necessary pretreatment is appropriately installed in the cell culture apparatus. Cell seeding can be appropriately performed by a known method or a method analogous thereto. For example, the cells may be suspended in the culture solution and dropped onto the cell culture support with a pipetteman or the like. The number of cells can be appropriately determined according to the type of cells to be cultured and the size of the cell sheet to be produced. In the step of culturing cells, a flow may be generated in the culture solution. By creating a flow in the direction parallel to the main surface in the vicinity of the main surface of the cell culture support, it is possible to prevent excessive cells from stacking to form a colony, and to form a single-layer cell sheet. It can be obtained in a short time.

  The flow of the culture solution may be continuously generated during the step of culturing the cells, or may be intermittently generated. For example, the cell culture support can be placed in a culture vessel, the medium can be continuously supplied to and discharged from the culture vessel, and a flow can be formed in the culture vessel. Moreover, after seed | inoculating a cell, it may leave still without generating a flow for a while, and may generate | occur | produce a flow intermittently after that. By installing the cell culture support in a state of being floated in the culture solution, all the necessary nutrients can be taken from the culture solution and waste products can be discharged, so that the cells can be maintained in a good state.

  Further, in the method for producing a cell sheet according to the present invention, depending on the type of cell, it is not necessary to pass the cell, that is, to transfer to another container, and a cell culture support is installed in the cell culture device as necessary. The culture solution may be exchanged or circulated as it is. According to the cell culture support having a planar mesh structure, the cells repeat division even after the cell sheet is formed, and dead cells and deteriorated cells naturally fall off the cell culture support. Thus, the cell sheet can be cultured and maintained for a long time.

  As a specific apparatus for realizing the cell culture as described above, the apparatus shown in FIGS. 3 and 11 may be mentioned. In the apparatus of FIG. 3D, it has one reaction plate and a culture vessel, and at least one groove leading to the inside of the culture vessel is cut in the plate. Examples of the reaction plate include glass, resin, or metal plate. As the culture tank, a glass, resin, or metal cylindrical container may be used, and it is preferable to have a secret structure in which water does not leak by contacting the reaction plate while the bottom is opened. A cell culture support having a planar mesh structure is installed at a substantial center of the culture tank so as to float from the bottom of the culture tank. “Substantially centered” means that the radius may deviate by about 50% from the center. The culture tank is filled with a culture medium (culture solution) so that the support is immersed. The groove of the plate extends to a planar mesh structure, through which the culture solution is supplied from the lower part of the planar mesh structure. A tube is provided at the top (upper part) of the culture tank above the planar mesh structure, and the culture solution is discharged therefrom. By supplying and discharging in this manner to form a flow in a direction perpendicular to the opening (main surface) of the cell culture support, the planar mesh of the cell culture support is removed while removing waste products from the culture system. Cells can be cultured on the structure.

  The apparatus of FIG. 3H has one reaction plate, and a culture tank including a recess or an opening for storing a culture medium (culture solution) is formed at the approximate center of the plate. The approximate center has the same meaning as described above, and the same applies to the following. A cell culture support having a planar mesh structure is installed in the approximate center of the culture tank. Two or more grooves extending radially from the culture tank are cut in the reaction plate, and the grooves serve as a flow path to supply and discharge the culture solution and continue feeding fresh culture solution to the culture vessel. it can. In the present embodiment, the flow in the culture tank is formed in parallel along the opening (main surface) of the cell culture support. Examples of the material for the reaction plate include glass, resin, and metal plates. By creating a flow of the culture solution in this way, cells can be cultured on the planar mesh structure while removing waste products.

  In the apparatus of FIG. 11, it has two or more reaction plates, and at least one of the plates has a culture tank consisting of a depression or an opening for storing the culture medium. A cell culture support having a planar mesh structure can be installed, and each reaction plate has one or more grooves extending radially from the culture tank, and each groove has a flow path. No. Culture on the planar mesh structure of the cell culture support while feeding and discharging from the upper and lower plate grooves in the same medium, different medium, or a combination of medium and gas to promote different changes in the upper and lower cells. can do.

  In an example of the present invention, a cell culture support having a planar mesh structure can be disposed in the center of the above-described apparatus, and three or more radial flow paths are formed toward the center support. The medium can be supplied and discharged through the three or more radial channels.

(Medium (culture solution))
As the medium components, those usually used for culturing cells of this kind can be appropriately used. The medium may be a solid medium or a liquid medium, but is preferably a liquid medium (culture solution). As a component of a culture medium, the solution containing salts, a carbon source, and a nitrogen source is mentioned, for example. A buffering agent for maintaining an osmotic pressure and pH suitable for cells may be used. Examples of the carbon source or nitrogen source include glucose, vitamins, amino acids, hormones and the like. These components may be a natural medium composed of natural substances such as serum and tissue extract, a semi-synthetic medium composed of natural substances such as amino acids and salts, or a synthetic medium composed of known chemical substances. . Of these, a natural medium or a semi-synthetic medium using serum or the like is preferable. As the serum, heterologous serum and allogeneic serum can be used. The heterologous serum means serum derived from a different species of organism from the recipient when the cell culture is used for cell medicine or the like. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum. “Allogeneic serum” means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autoserum (also called autologous serum), ie, serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient.

Examples of the buffer include phosphate, sodium bicarbonate, carbon dioxide gas and the like.
The medium may contain metabolic inhibitors and antibiotics. Examples of antibiotics include penicillin, streptomycin, gentamicin and the like.

  Specifically, 10% fetal bovine serum as serum, DMEM (Dulbecco's modified Eagle medium) (GIBCO), DMEM high glucose (SIGMA), 20% fetal bovine serum (FBS), 0 using penicillin and streptomycin as antibiotics .1 mM NEAA (non-essential amino acid) (GIBCO), 100 units / mL penicillin, MEM (Eagle minimum essential medium) (SIGMA) containing 100 μg / mL streptomycin (GIBCO), and the like.

The culture conditions may be general conditions for culturing this type of cell, but the culture temperature is preferably 25 ° C. or higher and 50 ° C. or lower, more preferably 30 ° C. or higher and 45 ° C. or lower. More preferably, the temperature is 35 ° C. or higher and 40 ° C. or lower. For example, 5% carbon dioxide (CO ₂ ) is preferably supplied into the incubator.

(Hepatocytes, intestinal cells)
In the present invention, hepatocytes or intestinal cells were employed as cells to be cultured. As the hepatocytes, for example, primary cells collected from a living body, hepatocytes derived from iPS cells or ES cells, hepatoma cells such as HepG2 cells and HepaRG cells (human hepatoma-derived cells) can be used. HepG2 cells and HepaRG cells are preferred from the standpoint of established hepatocytes that have relatively little hepatocyte function such as transporter, metabolic activity, and albumin production and a small lot difference. The said hepatocytes may be used individually by 1 type, and may use 2 or more types together.

  As intestinal cells, cells that can differentiate into intestinal epithelial cells such as the small intestine and large intestine, or cells that have differentiated into intestinal epithelial cells can be used. Primary cells collected from a living body, intestinal cells derived from iPS cells or ES cells may be used. Intestinal cells may also be cancer cells such as Caco-2 cells (derived from human colon cancer). The said enteric cell may be used individually by 1 type, and may use 2 or more types together.

(Cell sheet, cell structure)
The present invention further relates to a cell sheet including a member having a planar mesh structure whose opening has a size through which at least one cell can pass, and hepatocytes or intestinal cells.
The cell sheet of the present invention can be produced by the method of producing a cell sheet according to the present invention described above.
The cell sheet obtained by the production method of the present invention may include a cell culture support. Since such a cell sheet is produced without going through the process of peeling from the container, it is not damaged by peeling. Also, depending on the cells, dead cells and deteriorated cells are automatically dropped during the manufacturing process, so that they are formed only from cells in good condition, and are high in quality and safety. In addition, it has excellent storage stability and is easy to transport. If the cell culture support is formed of a biocompatible material or a biodegradable material, it can be used for transplantation as it is.
The cell sheet provided by the present invention can be stored frozen.
For example, it is assumed that a cell sheet manufactured by the method for manufacturing a cell sheet of the present invention is sold as a product to a user who performs drug discovery such as a pharmaceutical company. When selling to users, cell sheets will be transported, and in such cases, cryopreserved cell sheets can be transported.

  It is also possible to seed and amplify different types of cells on at least one surface of the cell sheet to produce a higher order cell structure having a heterogeneous cell binding surface. Such a structure is useful for various basic researches and regenerative medicine, and can also be used for screening drug candidates.

  In addition, since the cell sheet according to the present invention can freely control strength and flexibility, a cell sheet having appropriate strength and flexibility can be produced and deformed or laminated to obtain a desired sheet. A cell structure having a shape and a function can also be produced. As an example, a cell sheet is rolled to produce a blood vessel-like structure.

  In addition, cell sheets can be laminated, or other types of cells can be cultured in the form of cell sheets. Cell structures can be produced from such sheets, and models and pathological models that mimic tissues and organs in the body. It is also possible to do.

  In addition, as described above, a cell sheet having a desired orientation and ordered structure can be produced by imparting shape anisotropy to the mesh structure. Such a cell sheet can be applied to, for example, regeneration of myocardial tissue having cell orientation.

  In a preferred embodiment of the present invention, intestinal cells can be cultured (co-cultured) together with fibroblasts. Especially, the co-culture form of Tig-1-20 cells (fibroblasts) / Caco-2 cells is particularly preferable. Such co-culture can greatly improve cell extension, and depending on the culture conditions and the culture period, for example, the results of transepithelial electrical resistance measurement values can be improved 3 to 5 times. be able to. In addition, a cell sheet having strong cell-cell adhesion can be produced by the above-described co-culture, and such a cell sheet having strong cell-cell adhesion is useful in a drug permeation test. That is, the absorption of the drug in the intestinal tract can be evaluated using the cell sheet of the present invention.

(Reduction of freezing and thawing damage)
According to a preferred embodiment of the present invention, damage caused by freezing and thawing on cells can be reduced. Although the freezing temperature of a cell is not specifically limited, For example, it freezes at -50 degreeC--100 degreeC. Melting may be performed at 25 ° C to 45 ° C. Although the cryopreservation period is not particularly limited, it can be preserved over a wide period from several days to several years. According to a preferred embodiment of the present invention, even in such cryopreservation, a good cell state is maintained after thawing, which is suitable for cell transport to the field of regenerative medicine / drug discovery, research use, etc. Application becomes possible.

(Improved marker gene expression level)
According to a preferred embodiment of the present invention, the expression of a mature liver marker gene in hepatocytes (eg, HepG2) is significantly increased. Examples of human liver maturation markers include albumin and the drug metabolizing enzyme CYP1A2. In addition, the comparison of the expression level of a gene can be standardized using the expression level of (beta) -actin. The rate of increase in the expression level of the marker gene varies depending on the culture conditions and the culture period, but 105% or more, 110% or more, 120% or more, or 150%, assuming that the culture time is 100% when cultured in a well or as a spheroid. The above is preferable.

  Also, according to a preferred embodiment of the present invention, the expression of the mature human intestinal marker gene in intestinal cells (eg, Caco-2) is significantly increased. Specifically, the expression of the transporter gene, tight junction related gene, and drug metabolizing enzyme gene is significantly increased. In addition, the comparison of the expression level of a gene can be standardized using the expression level of (beta) -actin. The rate of increase in the expression level of the marker gene varies depending on the culture conditions and the culture period, but it is preferably from 105 to 200% or more when the micromesh alone (non-perfusion type) is taken as 100%.

  The present invention will be described more specifically with reference to the following examples, but the present invention should not be construed as being limited thereby.

<Production example of planar mesh structure>
As a metal micromesh manufacturing process, first, a photoresist is applied on a substrate. The photoresist on the substrate was exposed to ultraviolet rays using a photomask. By developing the photoresist, the portion not exposed to ultraviolet rays was removed. A metal was adhered to the groove formed by removing the photoresist by electroforming (electroforming). After peeling the photoresist, the electroformed sheet (metal micromesh) was peeled from the substrate. Nickel was used as the metal.

<Method of micromesh culture>
FIG. 1 shows a micromesh culture method. Using a Kapton (registered trademark) tape, the micromesh sheet 2 was fixed on the silicon plate 1 having a hole having a diameter of 4 mm. Using a laboratory coater PDS-2010 (Parylene Japan), a metal micromesh 2 / silicon plate 1 was vapor-coated with Parylene (DPXC, CAS No. 28804-46-8) (Parylene Japan) excellent in biocompatibility. For the purpose of increasing the degree of cell adhesion to the micromesh, the parylene-coated micromesh 2 / silicon plate 1 was hydrophilized using a plasma cleaner (HARRICK PLASMA). After sterilizing the micromesh 2 / silicon plate 1 by ultraviolet irradiation, 50 μL of a cell suspension of 2 × 10 ⁶ cells / mL (total 1 × 10 ⁵ cells) was placed on a micromesh circle (about 0.126 cm ² ) having a diameter of 4 mm. Inoculation was carried out using pipette 3, and culture was started. The micromesh 2 / silicon plate 1 was placed on the stage 4 with the lower plate 5. The culture was performed by immersing it in the culture solution 6. In addition, the stage 4 has a form that does not block the opening of the planar mesh structure (FIG. 1D).
FIG. 2 shows micrographs of micromesh culture using two types of micromesh sheets A and B having different eye sizes and shapes. As the cells, HepG2 cells (postoperative) were used. All the results of the micromesh culture shown in the examples are the results obtained using the mesh sheet of the mesh size of the micromesh sheet A.

<Method for producing two types of perfusion devices>
The perfusion apparatus was prepared using PDMS (polydimethylsiloxane) (trade name: SILPOT 184, Toray Dow Corning). Each part was produced by pouring the PDMS solution which mixed the main ingredient and the hardening | curing agent by 10: 1 into the type | mold, and heat-processing after that. The mold was produced by modeling with a 3D printer AGILISTA-3200 (Keyence) and then coating with parylene. A syringe pump (kd Scientific) was used as a device for feeding the culture medium from the silicon tube. As a feature of each perfusion device, the block-type perfusion culture device (A to D in FIG. 3) is easy to remove the lid on the apparatus, so that the micromesh 2 / silicon plate 1 (stage 4) can be easily taken in and out. is there. However, although cell observation from the lower surface is possible, cell observation from the upper surface is difficult, and the number of PDMS parts required for device fabrication is large. On the other hand, the chip-type perfusion device (EH in FIG. 3) does not take time to produce because it has one PDMS part. Furthermore, not only cell observation from below, but also cell observation from the upper surface using, for example, a stereomicroscope can be easily performed. As a disadvantage, when the micromesh 2 / silicon plate 1 (glass plate 4) was taken in and out, a part where the PDMS device had to be peeled off from the substrate was considered. In the figure, 7 is a tube, 8 is a pedestal, and 9 is a floor board.

<Example 1> (FIG. 4)
Microhepatic culture / materials and method of human hepatoma cell line (HepG2 cell) Cell acquisition and maintenance HepG2 cell (Product No. RCB1886) was obtained from RIKEN BioResource Center (RIKEN BRC). For cell culture, DMEM (Dulbecco's Modified Eagle Medium) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin and 100 μg / mL streptomycin (GIBCO) was used. The cells were cultured in an incubator under conditions of 37 ° C. and 5% carbon dioxide (CO ₂ ).

Micromesh culture ^Five 1 × 10 ⁵ HepG2 cells were seeded on a micromesh sheet having a diameter of 4 mm and cultured for 5 days. The medium was changed every 2 days at 2 mL / well / time. The cells seeded on the micromesh were perfused using a stationary culture (2 mL medium / well) or a block perfusion device (FIGS. 3A-D) in the wells of a 12-well plate. The medium was perfused at a flow rate of 1 mL / day. As an independent experiment, an experiment in which the culture was performed for a total of 10 days was similarly performed. For comparison, cells were seeded and cultured on a well of a 12-well plate (IWAKI) or a 256-well agarose gel plate placed in a well of the 12-well plate. A 256-well agarose gel plate was produced using MicroTissues 3D Petri Disc micro-mold spheroids (size S, 16 × 16 array, fits 12 well plates) (product number Z764000-6EA, SIGMA-ALDRICH). In these culture systems (excluding the perfusion culture system), the medium was changed every two days in the same manner as the above-described micromesh culture system.

Gene Expression Analysis RNA extraction from HepG2 cells was performed using RNeasy Micro Kit (QIAGEN), and cDNA was synthesized using the extracted RNA and RiverTra Ace qPCR RT Kit (Toyobo). According to the above method, cDNA was obtained from cells cultured for 5 days or cells cultured for 10 days. Gene expression analysis was performed using the obtained cDNA sample, DNA primer, Power SYBR Green PCR Master Mix (APPLIED BIOS SYSTEMS), and Quant Studio 5 Real-Time PCR System (APPLIED BIOS SYSTEMS). The gene expression levels of albumin and drug-metabolizing enzyme CYP1A2, which are known as human liver maturation markers, were analyzed. The expression level of each gene was normalized using the expression level of β-actin known as a housekeeping gene. Values are mean ± SE (N = 3).

-Results FIG. 4 shows the results of gene expression analysis. Compared with culturing HepG2 cells on normal wells (W), gene expression levels of albumin and CYP1A2 are higher when spheroid (S) or micromesh (M) (stationary) is cultured. it was high. Furthermore, the expression of these genes was further increased by performing micromesh culture under medium perfusion (D). The conditions under which micromesh culture was performed under medium perfusion for 10 days were the conditions that most promoted the above gene expression in this experiment.

<Example 2> (FIG. 5)
Perfusion culture of HepG2 cells using two types of perfusion devices and their effects on gene expression / materials and methods HepG2 cells are cultured in a block-type perfusion device or a chip-type perfusion device in the same manner and conditions as in Example 1. did. However, unlike Example 1, the culture period was 7 days in this experiment. See Example 1 for methods from RNA extraction to gene expression analysis. Values are mean ± SE (N = 3).
-Results FIG. 5 shows the results of gene expression analysis. There was no significant difference in the expression levels of the albumin gene and CYP1A2 gene in HepG2 cells between the culture systems using the block-type perfusion device (B) or the chip-type perfusion device (C).

<Example 3> (FIG. 6)
Micromesh culture / material and method of human intestinal tract-derived cell line (Caco-2 cell) Cell acquisition and maintenance Caco-2 cell (Product No. RCB0988) was obtained from RIKEN BioResource Center (RIKEN BRC). For cell culture, MEM containing 20% fetal bovine serum (FBS), 0.1 mM NEAA (non-essential amino acid) (GIBCO), 100 units / mL penicillin, 100 μg / mL streptomycin (GIBCO) (Eagle minimum) Essential medium) (SIGMA) was used. The cells were cultured in an incubator under conditions of 37 ° C. and 5% carbon dioxide (CO ₂ ).

Micromesh culture ^Five 1 × 10 ⁵ Caco-2 cells were seeded on a micromesh having a diameter of 4 mm and cultured for 7 days. Cells seeded on the micromesh were statically cultured (2 mL medium / well) in the wells of a 12-well plate (IWAKI). For comparison, cells were seeded in wells of a 12-well plate. The medium was changed every two days.

Gene expression analysis RNA was extracted from Caco-2 cells using RNeasy Micro Kit (QIAGEN), and cDNA was synthesized using the extracted RNA and RiverTra Ace qPCR RT Kit (Toyobo). Gene expression analysis was performed using the obtained cDNA sample, DNA primer, Power SYBR Green PCR Master Mix (APPLIED BIOS SYSTEMS), and Quant Studio 5 Real-Time PCR System (APPLIED BIOS SYSTEMS). The expression level of genes highly expressed in mature human intestinal tissue was analyzed. The expression level of each gene was normalized using the expression level of β-actin known as a housekeeping gene. Values are mean ± SE (N = 3).

Results FIG. 6 shows the results of gene expression analysis in Caco-2 cells. Compared to the case of culturing Caco-2 cells in normal wells, when the mesh mesh culture is performed, tight junction-related genes (ZO-1 gene, Occludin gene, Claudin-1 gene, Claudin-4 gene, The expression levels of Claudin-7 gene, Tricellulin gene), transporter gene (BCRP gene, OATP1A2 gene, PEPT1 gene), and drug metabolizing enzyme gene (CYP1A2 gene) increased.

<Example 4> (FIGS. 7 and 8)
Production / materials and method of PDMS micromesh In order to observe cells subjected to micromesh culture under a fluorescence microscope, a micromesh with extremely small autofluorescence was produced using PDMS. As a production procedure, first, a PDMS solution (Toray Dow Corning) in which a main agent and a curing agent were mixed at a ratio of 10: 1 was prepared. The viscosity of the PDMS solution was reduced by mixing the PDMS solution and a solvent (for example, hexane [Wako Pure Chemicals]) at a ratio of 1:12. The adjusted PDMS / hexane solution was applied to a metal micromesh at 6000 rpm for 10 seconds using a spin coater MS-B100 (Mikasa), and heat-treated to cure PDMS (one PDMS coating). In order to increase the strength of the micromesh, the PDMS / hexane solution was applied again (8000 rpm, 5 seconds) and heat-treated (PDMS coating twice). The metal part of the micromesh was etched with 0.1 N HCl solution. After washing, it was dried to complete a PDMS micromesh (FIG. 7). Plasma treatment was performed about 1-2 hours before cell seeding.

The culture and microscopy, materials and methods PDMS micromesh using PDMS micro mesh Caco-2 cells were plasma treated and plated 1X10 ⁵ cells of Caco-2 cells on the mesh diameter circle 4 mm. A sheet having a thickness of about 0.1 to 0.2 mm was used to fix the mesh. Cell culture was performed in the same manner as in Example 3. On the 4th day of culture, in order to stain the cell membrane, a medium containing 0.05 mg / mL DiI (1,1 ″ -dioctadecyl-3,3,3 ′, 3′-tetramethyllindocarbocyanine perchlorate) at 37 ° C. for 20 minutes Processed. After washing, the cells were fixed with 4% paraformaldehyde (PFA), and after washing, the cell / PDMS micromesh sheet was covered with a mounting medium VECTTASHIELD (VECTOR) containing DAPI (4 ′, 6-diamidino-2-phenylindole). And fixed between glass slides. The cell membrane (DiI) and cell nucleus (DAPI) were observed with a confocal microscope LSM 800 (ZEISS). The results are shown in FIG.

<Example 5> (FIGS. 9 and 10)
Measurement of transepithelial electrical resistance (TEER) of Tig-1-20 cells / Caco-2 cell sheets prepared by micromesh culture
-Materials and Methods Fetal lung-derived normal fibroblast Tig-1-20 cells (Product No. JCRB0501) were obtained from JCRB Cell Bank (National Research and Development Corporation, Pharmaceutical Substrate, Health and Nutrition Laboratory). IMatrix-511 Silk (Nippi) was coated on a 4 mm diameter micromesh circle of a PDMS device for transepithelial electrical resistance measurement (FIG. 9), and 1 × 10 ⁵ Tig-1-20 cells were seeded. Two days after seeding, 1 × 10 ⁵ Caco-2 cells were seeded on a Tig-1-20 cell sheet. As a target group, a transepithelial electrical resistance measurement device in which Tig-1-20 cells (with iMatrix-511 Silk) or Caco-2 cells (without iMatrix-511 Silk) were seeded was prepared. In the case of this example, the whole or about 1/3 of the medium was replaced with a fresh medium twice a week. The culture conditions other than the above medium exchange method and frequency are the same as in Example 3. Transepithelial electrical resistance was performed using EVOM2 (WPI) and EVOM2 electrode STX2 (WPI). Co-culture of Tig-1-20 cells / Caco-2 cells (N = 3, mean ± SE), Tig-1-20 cells alone (N = 3, mean ± SE), Caco-2 cells (N = 5- 6, mean ± SE). In addition, 8 in a figure is a base with a cylindrical wall.

-Results FIG. 10 shows the results of transepithelial electrical resistance measurement values in Tig-1-20 cell (fibroblast) / Caco-2 cell co-culture, Caco-2 cell single culture, or Tig-1-20 cell single culture. Show. There was no significant change in transepithelial electrical resistance value between Tig-1-20 cell culture alone and Caco-2 cell culture alone. This is presumably because Tig-1-20 cells, which are fibroblasts, are not highly tightly bound. In addition, in the case of a cell sheet composed only of Caco-2 cells, it was considered that Caco-2 could not fill the micromesh eyes uniformly without gaps, and electricity flowed through the gaps. On the other hand, in the co-culture, a high electrical resistance value was measured from the day after the co-culture. From this result, it was inferred that Caco-2 cells formed a sheet without a gap on the cell sheet formed by Tig-1-20 cells. The electrical resistance value (about 80-140 Ω · cm ² ) shown by co-culture of Tig-1-20 cells / Caco-2 cells is the previously reported in vivo large intestine (300-400 Ω · cm ² ) or small intestine (50-100 Ω · cm ²⁾ was comparable to the electric resistance value of (Srinivasan, B., et al. , TEER measurement techniques for in vitro barrier model systems. J Lab Autom, 2015. 20 (2) : p. 107-26). This suggested the possibility that the Caco-2 cell group under co-culture acquired a function (for example, cell-cell adhesive force, etc.) partially similar to the intestinal cell group in the living body.

Vertical perfusion device (Figure 11)
If different liquids or gases can be flowed up and down the cell sheet produced using the micromesh sheet, for example, when reconstructing the lumen side (apical side) and basement membrane side (basal side) of the intestinal tract in a living body The micro mesh sheet was considered to be a useful tool. FIG. 11 shows a schematic diagram of a perfusion device in which the joint surfaces of the upper and lower flow paths are micromesh sheets. By using this device, for example, it was considered possible to evaluate drug permeability and moving direction with respect to a cell sheet prepared by micromesh culture.

<Example 6> (FIG. 12)
Morphological change and method after freezing and thawing of HepG2 cells cultured in micromesh or spheroid cultures ⁵ × 1 × 10 ⁵ HepG2 cells were seeded on a micromesh sheet having a diameter of 4 mm and cultured. In this experiment, cells were cultured with DMEM high glucose (SIGMA). For comparison, cells were seeded and cultured on a 256-well agarose gel plate placed in a well of a 12-well plate. Two days later, the medium was removed from the wells, cell preservation solution CELLBANKER1 (ZENOAQ) was added (2 mL / well), and the plate was left in a −80 ° C. freezer. After 4 hours, the plate was moved into an incubator at -80 ° C to 37 ° C. After about 50 minutes, it was confirmed that CELLBANKER1 was completely dissolved. CELLBANKER1 in the well was replaced with a medium, and the cell morphology after 2 days of culture was examined. In order to confirm changes in cell membrane stability corresponding to changes in cell morphology, 1 μg / mL PI (Propidium Iodide) was added to the medium used for the two-day culture.
Results The cell morphology after 2 days of freezing and thawing is shown in FIG. While the spheroid morphology was largely destroyed by culturing for 2 days after freezing and thawing, no significant morphological change was observed in the cell sheet under micromesh culture. It was suggested that micromesh culture may be a culture method with less freeze-thaw damage to cells compared to spheroid culture method.

  In the present invention, hepatocytes or intestinal cells are used. The origin of the cell is not particularly limited, but is preferably derived from a human. Rat primary cultured hepatocytes, which are normal hepatocytes advantageous in terms of function, can also be used, but it is desirable to use human cells because their functions, particularly metabolic enzymes, are completely different depending on the animal species.

・ Caco-2
As a small intestinal membrane model, the human colon cancer-derived cell line Caco-2 is preferably used. Caco-2 cells are recently attracting attention as a cultured cell line that can be evaluated for absorbability by the intestinal tract, and were isolated and established from human colon cancer in 1977. It is known that when Caco-2 cells are cultured in a monolayer, they show morphological and biochemical membrane polarity such as formation of villi and tight junctions, and differentiate into absorptive epithelial cells in the small intestine.

・ HepG2
HepG2 is a human liver cancer-derived cell line established by Aden et al. The advantage of using human cells is great. This cell line derived from human has a metabolic enzyme called cytochrome P450 (CYP) 1A1 / 2. In normal culture conditions, it has less than 1/10 of the activity in vivo, but the activity can be increased by adding a chemical substance (3-methylcholanthrene, etc.) having an inducing action to promote the production of this enzyme. Can be maximized. In vivo, hepatocytes are present in a very high density state, which is said to be greatly involved in the expression of liver function.

  Since Caco-2 cells and HepG2 cells are cancer-derived cells, they can proliferate indefinitely, so that a large number of cells in a uniform state can be obtained. In addition, because human-derived cells with the same gene sequence are used, convincing results can be obtained in assessing human health risk.

  All of the above points give suggestions for various developments including clinical development such as treatment and testing, and research and utilization in the field of biochemistry.

1 Silicon plate 2 Micromesh sheet 3 Pipette 4 Stage 5 Lower plate 6 Medium (culture solution)
7 Tube 8 Base 9 Floor plate 101 Glass substrate 102 Metal layer 103 Resist layer 105 Ultraviolet light (light)

Claims (17)

Culturing hepatocytes or intestinal cells using a cell culture support having a planar mesh structure, wherein the opening of the mesh structure has a size that allows at least one cell to pass through. A method for producing a cell sheet, comprising: The method for producing a cell sheet according to claim 1, comprising culturing the cell culture support in contact with the hepatocytes or intestinal cells. The method for producing a cell sheet according to claim 1 or 2, wherein the cell culture support is placed in a medium to culture the hepatocytes or intestinal cells. The method for producing a cell sheet according to any one of claims 1 to 3, wherein the hepatocytes or intestinal cells grow in the opening of the cell culture support. The method for producing a cell sheet according to any one of claims 1 to 4, wherein the cell culture support having a planar mesh structure is plate-like and has a thickness of 1 µm to 50 µm. The method for producing a cell sheet according to any one of claims 1 to 5, wherein the cell culture support having the planar mesh structure is plate-shaped and has a large number of polygonal openings in plan view. The method for producing a cell sheet according to any one of claims 1 to 6, wherein, in the cell culture support having the planar mesh structure, a width of the frame dividing the opening in a plan view is 1 µm or more and 50 µm or less. . The frame includes a plurality of substantially parallel straight lines extending in the horizontal direction, a straight line extending in the right diagonal direction between the two horizontal straight lines, and a left diagonal direction in plan view. A straight line forms a continuous broken line structure so as to pass between the two horizontal lines, and a triangle is formed by the horizontal line, the right diagonal line, and the left diagonal line. The method for producing a cell sheet according to claim 7, wherein a large number of triangular openings are arranged in the support body. The cell sheet production according to claim 8, wherein a large number of triangular openings formed in the support body are substantially equilateral triangles, and the length of one side of the substantially equilateral triangular openings is 30 µm or more and 500 µm or less. Method. The method for producing a cell sheet according to any one of claims 1 to 9, wherein the cell culture support having a planar mesh structure is a plate-like porous body made of metal or resin. The cell according to any one of claims 1 to 10, wherein the cell culture support is placed in a culture tank, and a medium is continuously supplied to and discharged from the culture tank to form a flow in the culture tank. Sheet manufacturing method. The method for producing a cell sheet according to claim 11, wherein an apparatus having any one of the following structures and functions (a) to (c) is used as an incubator for culturing the hepatocytes or intestinal cells.
(A) It has one reaction plate and a culture tank, and at least one groove leading to the inside of the culture tank is cut in the plate, and a planar mesh structure is formed at the approximate center of the culture tank. A cell culture support having a surface of the culture tank can be set to float from the bottom of the culture tank, the culture tank can be filled with a medium so that the support is immersed, and the planar mesh can be inserted through the groove of the plate. It is possible to culture on the planar mesh structure of the cell culture support while removing waste products by supplying the medium from the lower part of the structure and discharging the medium from the tube provided at the upper part of the culture tank. it can.
(B) It has one reaction plate, and a culture tank is formed at the approximate center of the plate, which is a depression or opening for storing the culture medium. A cell culture support having a structure can be installed, and the reaction plate has two or more grooves extending radially from the culture tank. The grooves serve as flow paths to supply and discharge the medium. By continuously feeding a fresh medium to the culture tank, it is possible to culture on the planar mesh structure of the cell culture support while removing waste products.
(C) It has two or more reaction plates, and at least one of the plates has a culture tank composed of a depression or an opening for storing the culture medium, and a planar shape at the approximate center of the culture tank. A cell culture support having a mesh structure can be installed, and each reaction plate is provided with one or more grooves extending radially from the culture tank. Cultivation on the planar mesh structure of the cell culture support while supplying and discharging from the groove of the plate in the same medium, different medium, or a combination of medium and gas to promote different changes in the upper and lower cells. it can. A cell culture support having the planar mesh structure can be disposed at the center of the apparatus, and three or more radial flow paths are formed toward the center support, and supply and discharge of the medium. The method for producing a cell sheet according to claim 12, wherein the step is performed through the three or more radial flow paths. The method for producing a cell sheet according to any one of claims 1 to 13, wherein intestinal cells are cultured together with fibroblasts using the cell culture support having the planar mesh structure. A cell sheet comprising a member having a planar mesh structure whose opening has a size through which at least one cell can pass, and hepatocytes or intestinal cells. A cell sheet produced by the method according to any one of claims 1 to 14. The cell sheet according to claim 15 or 16, which is stored frozen.