* NOTICE *
`
`JPO and INPIT are not responsible for any damages caused by the use of this translation.
`
`1. This document has been translated by computer. So the translation may not reflect the original precisely.
`
`2. **** shows a word which cannot be translated.
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`3. in the drawings, any words are not translated.
`
`Publication Number
`
`JP20191780354A
`
`Bibliography
`
`(19) [Publication country] JP
`
`(12) [Kind of official gazette] A
`
`(11) [Publication number] 2019180354
`
`(43) [Date of publication of application] 20191024
`
`(54) [Title of the invention] METHOD FOR PRODUCING CELL SHEET AND CELL
`
`SHEET
`
`(51) [International Patent Classification]
`
`C1i2N
`
`5/071
`
`(2010.01)
`
`[Ft]
`
`Ci2N
`
`5/071
`
`(21) [Application number] 2018078321
`
`(22) [Filing date] 20180416
`
`(71) [Applicant]
`
`[Name] INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH
`
`(72) [Inventor]
`
`[Full name] HORI TAKESHI
`
`[Full name] KUROSAWA OSAMU
`
`[Full name] [(WATA HIROO
`
`[Full name] TAN] NOBUTAKA
`
`{Theme code (reference)]
`
`4B065
`
`[F-term (reference) ]
`
`4BOS65AA90X
`
`4B065BC41
`
`4B065CA44
`
`

`

`4B065CA46
`
`Abstract
`
`(57) [Overview]
`
`PROBLEM TO BE SOLVED: 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 ceil 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
`
`Claim
`
`[Patent Claims]
`
`[Claim 1]
`
`A method of manufacturing a cell sheet comprising culturing a hepatocyte or an intestinal
`
`cell using a cell culture support having a planar mesh structure, wherein an opening of
`
`said mesh structure is sized to allow passage of at least 1 cells.
`
`{Claim 2]
`
`The method for producing a cell sheet according to claim 1, comprising culturing the cell
`
`culture support in a state in which the hepatocytes orintestinal cells are contacted.
`
`[Claim 3]
`
`The method for producing a cell sheet according to claim 1 or 2, wherein the cell culture
`
`support is placed in a culture medium to culture the hepatocytes cr intestinal cells.
`
`[Claim 4]
`
`A method for producing a cell sheet according to any one of claims 1 to 3, wherein said
`
`hepatocytes or intestinal cells grow at an opening of said cell culture support.
`
`[Claim 5]
`
`The methodfor producing a cell sheet according to any one of claims 7 to 4, wherein the
`
`cell culture support having the planar mesh structure is plate-shaped and has a thickness
`
`of 1 mor more and 50 u mor less.
`
`[Claim 6]
`
`The method for producing a cell sheet according to any one of claims 7 to 5, wherein the
`
`cell culture support having the planar mesh structure is a plate and has many polygonal
`
`openingsin plan view.
`
`

`

`[Claim 7]
`
`The method of manufacturing a cell sheet according to any one of claims 1 to 6, wherein
`
`in a cell culture support having a planar mesh structure, a width of a frame for partitioning
`
`an opening in a plan view is 1 p m or more and 50 um orless.
`
`[Claim 8]
`
`in this case, a plurality of straight lines extending in a horizontal direction and a straight
`
`line extending in a diagonal direction of the right side and extending in a diagonal
`
`direction of the right side, and a straight line extending in a diagonal direction of the left
`
`side are arranged betweena plurality of substantially parallel straight lines extending in
`
`a horizontal direction in a plan view, and a straight line structure is continuously formed
`
`between the 2 horizontal lines in the 2 lateral directions. The method of manufacturing a
`
`cell sheet according to claim 7, wherein a triangle is formed by a straight line in a
`
`horizontal direction, a straight fine in a right oblique direction, and a straight line in a left
`
`oblique direction, whereby a plurality of triangular openings are arranged in the support.
`
`[Claim 9]
`
`The method of manufacturing a cell sheet according to claim 8, wherein a plurality of
`
`triangular openings formed in the support body have an approximately equilateral
`
`triangle, and a length of one side of the opening of the substantially equilateral triangle
`
`is 30 p m or more and 500 p morless.
`
`{Claim 10]
`
`The methodfor producing a cell sheet according to any one of claims 1 to 9, wherein the
`
`cell culture support having the planar mesh structure is a metal or resin plate porous
`
`body.
`
`{Claim 114]
`
`A method for producing a cell sheet according to any one of claims 1 to 10, wherein the
`
`cell culture support is placed in a culture tank, and the culture medium is continuously
`
`supplied and discharged into the culture tank to form a flow in the culture tank.
`
`[Claim 12]
`
`The method for producing a cell sheet according to claim 11, wherein an apparatus
`
`having the structure and function of any one of the following (a} to (c) is used as an
`
`incubator for culturing said hepatocytes or intestinal cells.
`
`(a) 1 reaction plates and a culture tank, and at least one groove leading to the inside of
`
`the culture tank is cut into the plate, and a cell culture support having a flat mesh structure
`
`can be instailed in a substantially center of the culture tank so as to float from the bottom
`
`of the culture tank. The culture tank can be filled with a medium such that the support
`
`body is immersed in a medium, and the culture medium is supplied from a lower part of
`
`

`

`the planar mesh structure through a groove of the plate, and a tube provided at an upper
`
`part of the culture tank is provided.
`
`By draining the medium, it can be cultured on a planar mesh structure of the cell culture
`
`support while removing waste products.
`
`(b) a culture tank comprising 1 reaction plates, the culture tank comprising a recess or
`
`an opening in which a culture medium is stored ; A cell culture support having a planar
`
`mesh structure can be placed at substantially the center of the culture tank, and 2 or
`
`more grooves extending radially from the culture tank are cut into the reaction plate, and
`
`the groove forms a channel and supplies and discharges the medium. By continuing to
`
`feed fresh media into the culture vessel, a planar mesh structure of the cell culture
`
`support can be cultured while removing waste products.
`
`(c) a culture tank comprising 2 or more reaction plates, wherein the culture tank
`
`comprises a recess or an opening in which a medium is stored at least approximately in
`
`the center of at least one of the plates, and a cell culture support having a planar mesh
`
`structure can be placed at substantially the center of the culture tank ; In each of the
`
`reaction plates, one or more grooves extending radially from the culture tank are cut in
`
`each plate, and the grooves are supplied and discharged from the grooves of the upper
`
`and lower plates by supplying and discharging the same medium, a different medium, or
`
`a combination of the medium and the gas. it can be cultured on a planar mesh structure
`
`of the ceil culture support while promoting different changes in and on the cell.
`
`[Claim 13]
`
`A method of producing a cell sheet according to claim 12, wherein a cell culture support
`
`having a planar mesh structure can be disposed in a center of the device, and 3 or more
`
`radial channels are formed toward the central support, and the supply and discharge of
`
`the medium is performed through the 3 or more radial channels.
`
`[Claim 14]
`
`A method for producing a ceil sheet according to any one of claims 1 to 13, wherein the
`
`cell culture support having the planar mesh structure is used to culture intestinal cells
`
`together with fibroblasts.
`
`[Claim 15]
`
`A cell sheet comprising a member having a planar mesh structure, wherein the opening
`
`is sized to allow passage of at least 1 cells, and hepatocytes orintestinal cells.
`
`[Claim 16]
`
`A cell sheet produced by the method of any one of claims 1 to 14.
`
`{Claim 17]
`
`

`

`A cell sheet according to claim 15 or 16, which is stored frozen.
`
`Description
`
`{Detailed description of the invention]
`
`[Technical field]
`
`{0001}
`
`The present invention relates to a method for producing a cell sheet using hepatocytes
`
`or intestinal cells, and a cell sheet.
`
`[Background of the Invention]
`
`{[O002]
`
`it has been attempted to apply to various medical and drug discovery technologies by
`
`using cultured cells to produce higher ceil structures. As a representative example
`
`thereof, various kinds of applications of cell sheets, medical and drug discovery are
`
`applicable. A cell sheet is typically a sheet-like cell aggregate of at least a monolayer
`
`formed by mutual bonding of adjacent cells. Cell sheets have begun to be widely used
`
`in fields such as regenerative medicine and drug discovery.
`
`{C003}
`
`On the other hand, it is not easy to produce a good quality cell sheet. Conventionally, a
`
`method of inoculating a target cell into a container such as a Petri dish and growing it
`
`has been used. However, when the numbercf cells increases and becomes confluent
`
`(very close and dense), contact inhibition (contact inhibition) occurs, so that a passage
`
`is required, resulting in trouble and cost. in some cases, the above problems may have
`
`a profound impact on the quality of the cell sheet.
`
`[0004]
`
`In a method of culturing using a container such as a Petri dish, since cells adhere to a
`
`container,
`
`it
`
`is necessary to peel off the cell from the container.
`
`In particular,
`
`it
`
`is
`
`necessary to add mechanical or chemical manipulations to recover the cell sheet, such
`
`as when the cell is tightly bound to the container. Further, when a cell sheet is used for
`
`regenerative medicine and drug discovery, quality is
`
`important. However,
`
`in the
`
`conventional method of producing a cell sheet on the surface of a container such as a
`
`Petri dish, a cell sheet cannot be selectively removed, and only a cell sheet containing a
`
`mixture of
`
`live and dead cells can be produced. Further, as described above,
`
`deterioration of cells due to contact inhibition also occurs, and thus further improvement
`
`has been required in order to adapt to regenerative medicine and drug discovery.
`
`fO005}]
`
`

`

`in response to such problems, i has been proposed to use a cell culture support having
`
`a cell culture support having a planar mesh structure, wherein an cpening of a mesh
`
`structure is larger than a cell to be cultured (Patent Document 1). In this case, a mouse
`
`embryonic fibroblast (MEF), a human fetal
`
`lung fibroblast (TIG 120), and a mouse
`
`embryonic stem cell (ES) are cultured. When cells are seeded in a state in which these
`
`cell culture supports are suspended in a culture medium, the cells spontaneously extend
`
`into the openings of the mesh structure, and the ceils in contact with each other adhere
`
`to each otherto fill the openings of the mesh structure, thereby forming a monolayerof
`
`cell sheets. Even if
`
`the cells proliferate, dead cells and cells with poor state
`
`spontaneously fall out of the cell culture support, so that the culture can be continued for
`
`a long time without performing passage. As a result, a highly safe cell sheet consisting
`
`only of viable cells is obtained. Further, it is considered that the strength and flexibility of
`
`a cell sheet can be increased by adjusting an angle and the like in an incubator.
`
`{CO06]
`
`it has also been reported that trophoblast cells are induced by culturing iPS celis using
`
`a mesh (Non-Patent Document 1).
`
`[C007]
`
`In drug discovery,
`
`it
`
`is recognized that mature human celis are used in ADME test
`
`(absorption, distribution, metabolism, excretion test) and toxicity test, but it is still difficult
`
`to stably obtain and culture such functional human cells.
`
`[Prior art reference]
`
`[Patent document]
`
`{0008}
`
`{Patent document 1]international Publication
`
`No. WO2015/005394 pamphlet
`
`[Non-patent documents]
`
`[0009]
`
`[Non-patent document 1]Okeyo KO et al. Tissue Eng Part C Methods. 2015
`
`Oct;21(10):1105-15.
`
`{Non-patent document 2]Soldatow, V.Y., et al., In vitro models for liver toxicity testing.
`
`Toxicol Res (Camb), 2013. 2(1): p. 23-39.
`
`[Summaryof the invention]
`
`[Problem to be solved by the invention]
`
`[0010]
`
`Experimental animals, normal human hepatocytes, intestinal cells, human hepatocytes,
`
`and the like have been usedin orderto evaluate the toxicity of chemical substances such
`
`as new medicines and the like and in vivo kinetics. For animal experiments, it has been
`
`

`

`found that there is a large species difference in the metabolism and excretion of
`
`chemicals, and it is recognized thatit is difficult to accurately obtain information relating
`
`to the toxicity, metabolism and excretion of humans from experimental animals. Further,
`
`as for normal human hepatocytes or intestinal cells derived from surgical patients and
`
`the like, there is a problem that individual differences are large and cannot be obtained
`
`stably at low cost. With regard to a human cell line, there is an advantage in that it is
`
`easy to grow and managein vitro (ex vivo), while it has a disadvantage in that it has a
`
`significantly reduced function as compared with normal cells in vivo.
`
`{001 14]
`
`To solve these problems, a human cell line is three dimensionally cultured.
`
`Studies have been actively conducted to close the state of cells in vivo. Although there
`
`is spheroid cultivation as a general three-dimensionai cell culture system, In order that
`
`oxygen and a nutrient may become being hard to spread round an internal cell group or
`
`the defect of becoming difficult to carry out internal microscopic observations may occur
`
`further as a spheroid (cell lump) enlarges, development of a new three-dimensional cell
`
`culture system is desired (nonpatentliterature 2).
`
`[0012]
`
`The system which cultures Caco-2 cell on polycarbonate membrane with the many open
`
`hole, etc.
`
`in order to evaluate the absorption from the small
`
`intestine of a sample
`
`compound, etc.
`
`is used. However,
`
`in this culture system, the Caco-2 ceils can be
`
`regarded as a 2 dimensional culture system basically because the cells can be adhered
`
`and extended flat on a perforated substrate, and therefore, it can be regarded as a 2
`
`dimensional culture system. Since such a culture system is not sufficient as the
`
`expression amount of the transporter in Caco-2 cell, there are not few cases where the
`
`moving state of a test compound cannot be evaluated correctly. In addition, since the
`
`porosity of the porous membrane commonly used for Caco-2 cells is around 15%, it is
`
`considered that the information on the transepithelial electrical resistance and the
`
`compound permeability obtained from the Caco-2 cell sheet on the porous membrane
`
`has a different property from thosein the living body 2. 2.
`
`[0013]
`
`To provide a method for producing a cell sheet containing a hepatocyte or an intestinal
`
`cell having a function closer to a living body.
`
`{Meansfor soiving the problem]
`
`[0014]
`
`The present inventors have conducted extensive studies to solve the problem that a large
`
`

`

`number of techniques such as those described in Non-Patent Document 2 have resulted
`
`in insufficient maturation of cells. As a result, a ceil culture support having a planar mesh
`
`structure is provided by culturing hepatocytes or intestinal cells using a cell culture
`
`support in which the openings of the mesh structure are sized to allow at least 1 cells to
`
`pass through. It has been found that a cell sheet containing a hepatocyte or an intestinal
`
`cell having unexpectedly high maturity can be produced, and thus the present invention
`
`has been compieted. According to the present invention, there is provided the following
`
`invention.
`
`{001 5]
`
`1> A methodfor producing a cell sheet comprising culturing a hepatocyte or an intestinal
`
`cell using a cell culture support having a planar mesh structure, wherein the opening of
`
`the mesh structure is sized to allow passage ofat least 1 cells.
`
`(2) The method for producing a cell sheet according to (1), comprising culturing the cell
`
`culture support in a state in which the hepatocytes or intestinal cells are in contact with
`
`each other.
`
`(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 at an 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 the planar mesh structure is a plate and has a thickness of 4
`
`4m or more and 50 p m orless.
`
`(6) The method for producing a cell sheet according to any oneof(1) to (5), wherein the
`
`cell culture support having the planar mesh structure is a plate, and has many polygonal
`
`openings in plan view.
`
`(7) The method for producing a cell sheet according to any one of (7) to (6), wherein in
`
`the cell culture support having the planar meshstructure, a width of a frame for sectioning
`
`an openingin a plan view is from 1 pm to 50 um.
`
`8> A straight line has a pofygonai structure in which a plurality of straight lines extending
`
`in a horizontal direction and extending in a horizontal direction and a straight line
`
`extending in a diagonal direction of a right side and extending in a diagonal direction of
`
`a right side and a straight line extending in an oblique direction obliquely toward the left
`
`side are arranged so as to pass betweenthe 2 straight lines in the horizontal direction,
`
`in a pian view ; and 2 A cell sheet according to (7), wherein a triangle is formed by a
`
`straight line in a horizontal direction, a straight line in a right oblique direction, and a
`
`straight line in a left oblique direction, whereby a large numberoftriangular openings are
`
`

`

`arranged in the support.
`
`REE
`
`(9) The method of manufacturing a cell sheet according to (8), wherein a plurality of
`
`triangular openings formed in the support have a substantially equilateral triangle, anda
`
`length of a side of an opening of the substantially equilateral triangle is 30 p m or more
`
`and 500 um or less.
`
`(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 metal or resin plate-like
`
`porous body.
`
`(11) The method for producing a cell sheet of a cell according to any one of (1) to (10),
`
`wherein the cell culture support is placed in a culture tank, and the culture medium is
`
`continuously supplied and discharged into the culture tank to form a flow in the culture
`
`tank.
`
`(12) The method for producing a cell sheet according to (11), wherein an apparatus
`
`having the structure and function of any one of the following (a) to (c) is used as an
`
`incubator for culturing the hepatocytes or intestinal cells.
`
`(a) 1 reaction plates and a culture tank, and at least one groove leading to the inside of
`
`the culture tank is cut into the plate, and a cell culture support having a fiat mesh structure
`
`can be installed in a substantially center of the culture tank in such a manner as to float
`
`from the bottom of the culture tank. The culture tank can be filled with a medium so that
`
`the support is immersed in the culture tank, a culture medium is supphed from a lower
`
`part of the planar mesh structure through a groove of the plate, and a medium is
`
`discharged from a tube provided at an upper part of the culture tank. Cells may be
`
`cultured on a planar mesh structure of the cell culture support while removing waste.
`
`(b) a culture tank comprising 1 reaction plates, the culture tank comprising a recess or
`
`an opening in which a culture medium is stored ; A cell culture support having a planar
`
`mesh structure can be placed at substantially the center of the culture tank, and 2 or
`
`more grooves extending radially from the culture tank are cut into the reaction plate, and
`
`the groove forms a channel and supplies and discharges the medium. By continuing to
`
`feed fresh media into the culture vessel, cells can be cultured on the planar mesh
`
`structure of the cell culture support while removing waste products.
`
`(c) a culture tank comprising 2 or more reaction plates, wherein the culture tank
`
`comprises a recess or an opening in which a medium is stored at least approximately in
`
`the center of at least one of the plates, and a cell culture support having a planar mesh
`
`structure can be placed at substantially the center of the culture tank ;
`
`In each of the
`
`

`

`reaction plates, one or more grooves extending radially from the culture tank are cut in
`
`each piate, and the grooves are supplied and discharged from the grooves of the upper
`
`and lower plates by supplying and discharging the same medium, a different medium, or
`
`a combination of the medium and the gas. Cells can be cultured on a planar mesh
`
`structure of the cell culture support while promoting different changes in and below the
`
`cell.
`
`(13) A method for producing a ceil sheet according to (12), wherein a cell culture support
`
`having a planar meshstructure can be placed in a center of the apparatus, and 3 or more
`
`radial channels are formed toward the central support, and the supply and discharge of
`
`the medium are performed via the 3 or more radial channels.
`
`(14) The method for producing a cell sheet according to any one of (1) to (13), wherein
`
`the cell culture support having the planar mesh structure is used to culture intestinal cells
`
`together with fibroblasts.
`
`15> A cell sheet comprising a member having a planar mesh structure, wherein the
`
`opening is sized to allow passage of at least 1 cells, and a hepatocyte or intestinal cell.
`
`(16) A cell sheet produced by the method according to any one of (1) to (74).
`
`(17) The cell sheet according to (15) or (16), which is stored in a frozen state.
`
`{Effect of the Invention]
`
`{0016}
`
`According to the present invention, by using a support having a planar mesh structure, it
`
`is possible to provide a cell sheet including hepatocytes and intestinal cells which are
`
`important in drug discovery.
`
`[Brief Description of the Drawings]
`
`{001 7]
`
`{[Fig. 1]FIG.
`
`1 a is a top view of a micromeshfixed with Kapton © tape, and partially
`
`enlarged view thereof ;. FIG. 1 b is a perspective view showing an embodiment in which
`
`cells are seeded on a micromesh. FiG. 1 c shows an embodiment of an incubator when
`
`a micromeshis installed ;
`
`it
`
`is a perspective view showing voice. FIG.
`
`1 d is a cross-sectional view of an
`
`embodiment of an incubator when a micromesh is installed ;.
`
`[Fig. 2JFIG. 2 is a microscopic image showing an embodiment of micromesh culture
`
`using HepG2cells in 2 types of mesh sizes A and B ; and 2.
`[Fig. 3]FIG. 3 ais a cross-sectional view of A “line showing a micromesh culture block
`
`perfusion device ;. FIG. 3 b is a top view (partially exploded view) showing a micromesh
`culture block perfusion device ;. FIG. 3 c is a cross-sectional view along line C ’
`
`

`

`showing a micromesh culture block perfusion device ;. FIG. 3 d is a perspective
`
`perspective view of a micromesh culture block perfusion device ;. FiG. 3 e is a cross-
`sectional view of an ’”
`line showing a micromesh culture chip perfusion device ;. FIG.
`
`3 f is a top view of a micromesh culture chip perfusion device ;. FIG. 3 g is a cross-
`sectional view taken along line G ’
`showing a micromesh culture chip perfusion
`
`device ;. FIG. 3 his a perspective perspective view of a micromesh culture chip perfusion
`
`device :.
`
`[Fig. 4]Fig.4 is a graph which showsthe relative expression amount of the liver mature
`
`marker gene in stock-ized hepatocyte HepG2 cell.
`
`[Fig. 5]FIG. 5is a graph showing the relative expression of fiver maturation marker genes
`
`in cultured hepatocyte HepG2 cells cultured using 2 types of perfusion devices ; and 2.
`
`[Fig. 6]FIG. 6 is a graph showing the relative expression of the intestinal maturation
`
`marker gene in the linearized intestinal cells Caco-2 cells (FIG. 2).
`
`{[Fig. 7]FiG. 7 is a microscope image showing a micro-mesh sheet made of PDMS.
`
`[Fig. 8]FIG. 8 is a microscopic image showing an embodiment in which Coco- 2 cells
`
`are cultured on a PDMS micromesh.
`
`[Fig. 9]FIG. 9 a is a cross-sectional view taken along fine A “of an apparatus for
`
`measuring transepithelial electrical resistance (TEER) of a cell sheet formed by
`
`micromesh culture. FIG. 9 b is a plan view of an apparatus for measuring transepithelial
`
`electrical resistance (TEER) of a cell sheet formed by micromesh culture. FIG. 9cisa
`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]FIG. 10 is a graph showing measurement of transepithelial electrical resistance
`
`(TEER) in a cell sheet formed by micromesh culture.
`[Fig. 11]FIG. 11 ais a cross-sectional view taken along line A ’
`
`showing a device for
`
`separately perfusing 2 kinds of culture solutions above and below a cell sheet formed by
`
`micromeshculture. FIG. 11 bis a plan view showing a device for separately perfusing 2
`
`kinds of culture solutions above and below a cell sheet formed by micromesh culture.
`
`FIG. 11 cis a perspective perspective view showing a device for separately perfusing 2
`
`kinds of culture solutions above and below a ceil sheet formed by micromesh culture ;.
`
`[Fig. 72]FIG. 12 is a microscope image comparing the difference between spheroid
`culture and micromesh culture.
`
`{[Fig. 13]FiG. 13 is a process explanatory diagram schematically showing an overview of
`
`a method of manufacturing a micromesh in a perspective view.
`
`[Fig. 14]JFiG. 14 is a process explanatory diagram schematically showing a part of a
`
`process of manufacturing a photomask using photolithography in a cross-sectional view.
`
`

`

`iFig. 15]Fig.15 is a shape of microscopic features (right figure: expansion) in which the
`
`culture results of HepG2 cell by micromesh are shown.
`
`{Mode for carrying out the invention]
`
`[0018]
`
`Embodiments of the present invention will be described below.
`
`in a method for producing a cell sheet of the present invention, a cell culture support
`
`having a planar mesh structure is used, and a cell culture support having a size in which
`
`at least 1 cells can pass through an opening of the mesh structure is used to culture a
`
`hepatocyte or an intestinal cell.
`
`[0019]
`
`The cell sheet formed by culturing cells using a cell culture support with a planar mesh
`
`structure is considered to be superior in oxygen / nutrient supply to cells because of
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`being thinner than a spheroid, and as a result, a cell with high maturity is expected to be
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`obtained. In addition, due to the feature of the cell sheet, it is possible to reconstruct a
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`layered structure of an in-vivo tissue. Furthermore, the cell sheet produced by micromesh
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`culture has an advantage that microscopic observation can be easily performed from
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`both the upper surface and the lower surface. Moreover, since the cell sheet is a cell
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`sheet having a small percentage of cell-cell adhesion, it may be useful for analysis of
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`permeability of a test substance. In this context, it is also possible to perfuse different
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`fluids up and down separately from the ceil sheet and is considered suitable for analysis
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`of drug permeability.
`
`[0020]
`
`(Cell Culture Support)
`Planar mesh structure
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`Cell culture supports that can be used in the present invention have a planar mesh
`structure.
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`By using a cell culture support having a planar mesh structure, it is possible to provide a
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`culture method which is less likely to freeze and damage to cells than a spheroid culture
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`method.
`
`[0021]
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`Each opening of the mesh structure is sized to allow for the passage of cells (at least 1
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`cells) to be cultured. A cell culture support may be placed in a culture medium to allow
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`cells to grow along their shape. As compared with a scaffold in which cells colonize a
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`scaffold larger in area than a cell, such as a scaffold, a cell spontaneously extends into
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`an opening of a mesh. Conventionally, a scaffold is used as a culture support at the
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`

`

`bottom of a uniform container or a scaffold having an opening smaller than that of a cell
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`such as a pore, so that the cell is fixed to the scaffold and grows to form a tissue.
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`Therefore, itis difficult to form a sheet only by adhesion betweencells. On the other hand,
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`when a support having a mesh structure having an opening larger than a cell is used,
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`cells spontaneously expand toward the center of the opening and adhere. As a result, a
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`single layer of cell sheet is easily formed so as to cover the opening. Note that, in the
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`present specification, the phrase "cell extends" or "cell grows" meansthat a cell deforms,
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`grows, or proliferates in a specific direction.
`
`{0022}
`
`Opening portion
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`in a planar mesh structure, a planar structure in which an opening of a predetermined
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`shape is regularly or irregularly repeatedly arranged regularly is preferred.
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`in a planar
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`mesh structure, it is more preferable to have many polygonal openings in plan view. An
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`opening of a given shapeis typically a regular polygon, such as an equilateral triangle,
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`a square, a regular hexagon, or the like, but may be a circle, an ellipse, or other polygon.
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`Note that, in this specification, the term "regular triangle", "square", "regular hexagon”,
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`and "reguiar polygon”is not limited to an equilateral triangle, a square, a regular hexagon,
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`and a regular polygon, and includes a substantially equilateral triangle, a substantially
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`square, a substantially regular hexagon, and a substantially regular polygon.
`
`{0023}
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`in addition, all of the openings need not be of the same shape, and a plurality of openings
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`may be provided as 1 sets of regularly or irregularly aligned planar structures.
`
`[0024]
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`A portion other than an opening of a planar mesh structure is called a frame or a frame
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`portion. in addition, a planar mesh structure may be referred to as a micromesh when
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`the opening portion has a micrometersize.
`
`[0025]
`
`An opening in a cell culture support having a planar mesh structure is sized to allow at
`
`ieast 1 ceils to be cultured to pass through. By a size through which at least 1 of the cells
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`in which an opening is cultured can pass is meant that the size of each of the opening
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`and of the cell is such that the cell can be passed through the opening with or without
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`deformation. The opening may be sized to allow cells to pass without contact with the
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`opening, or may be sized to be able to pass through and deform in contact with the
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`opening.
`
`it is good.
`
`

`

`fOO26}]
`
`An example in which at least 1 of the cells in which an openingis to be cultured is to be
`
`able to pass is a case wherethe area of the opening of the meshstructure is larger than
`
`the maximum area of the 1 cells at the time of seeding. A maximum area of 1 cells refers
`
`to an area when a cell
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`is cut so as to have a maximum cross-sectional area. The
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`maximum area of cells at the time of seeding means a maximum area in the state where
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`cells are seeded, |. e., before the cells adhere to the support and expand.
`
`[0027]
`
`in addition, a ceil culture support having a planar mesh structure may have a minimum
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`diameter of an opening larger than a maximum diameterat the time of seeding of a cell
`
`to be cultured. A minimum diameter of an opening may be appropriately measured by a
`
`person skilled in the art, and for example, in the case of a regular polygon, a length ofa
`
`straight line connecting 2 points on an edge through a center is a shortest straightline.
`
`A maximum diameter of a cell refers to a fength of a longest straight line of a straight line
`
`connecting 2 points around a cell.
`
`When there are 2 or more openings, the minimum diameter of the opening may be larger
`
`than the maximum diameter at the time of seeding of the cell to be cultured, or the
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`minimum diameter of the opening may be larger than the maximum diameter at the time
`
`of seeding of the ceil to be cultured at any of the openings.
`
`{0028}
`
`When a minimum diameter of an opening is larger than a maximum diameter of a cell,
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`when a cell culture support is placed horizontally and a ceil is seeded from above, there
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`is also a ceil that is fixed to a frame portion and a ceil that falls down without contacting
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`a frame. Only the cells fixed to the frame portion do notinitially fill the opening, but a cell
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`sheet is formed as a result of the cells spontaneously expanding toward the opening
`center.
`
`[0029]
`
`The cell culture support may have a minimum diameter of the opening of 2, 3, 4, 5, 7, 10,
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`or the like of the maximum diameter of the cell to be cultured.
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`In such a case, cells
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`seeded in the frame portion spontaneously extend to the opening portion, and the cells
`
`are connected te each other to fill the opening portion, thereby f