CELL CULTURE CHIP
`
`BACKGROUND
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`1. Technical Field
`
`[0001]
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`The present disclosure relates to a cell culture chip.
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`2. Description of the Related Art
`
`[0002]
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`Recently, an organ on a chip (OoC) has been actively developed as a
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`10
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`cell culture chip. (Refer to, for example, Japanese Patent Application No.
`
`2019-506861 and Japanese Patent Unexamined Publication No. 2019-180354).
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`The OoC is a cell culture chip that reproduces tissue functions in an organ on a
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`microscale by culturing cells in an artificial micro-space in which glass, resin,
`
`and the like are combined, as illustrated in FIG. 39 of Japanese Patent
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`15
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`Application No. 2019-506861.
`
`[0003]
`
`By adding drugto cells cultured by using the cell culture chip, tests
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`of the related art such as drug efficacy, toxicity test or absorption, metabolism,
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`excretion, and the like of the drug, which are evaluated by animal tests using
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`mice, can be performed in an artificial chip other than an organ.
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`20
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`SUMMARY
`
`[0004]
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`A cell culture chip according to one aspect of the present disclosure
`
`has a stack structure formed by sequentially stacking a first board provided
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`with a first electrode on a main surface, a first partition wall layer including a
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`25
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`first main flow path, and a first inlet flow path and a first outlet flow path
`
`connected to the first main flow path, a planar mesh structure sheet used as a
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`scaffolding material for cells, a second partition wall layer including a second
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`1
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`

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`main flow path, and a second inlet flow path and a second outlet flow path
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`connected to the second main flow path, and a second board provided with a
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`second electrode on a main surface, in which the planar mesh structure sheetis
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`sandwiched betweenthefirst partition wall layer and the second partition wall
`
`layer, among aperture ratios of a surface of the planar mesh structure sheet
`
`facing the first partition wall layer, an aperture ratio of a portion facing the
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`first main flow path is greater than an aperture ratios of portions facing the
`
`first inlet flow path and thefirst outlet flow path, and among apertureratios of
`
`a surface of the planar mesh structure sheet facing the second partition wall
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`10
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`layer, an aperture ratio of a portion facing the second main flow pathis greater
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`than an aperture ratios of portions facing the second inlet flow path and the
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`second outlet flow path.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`15
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`[0005]
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`FIG. 1A is an exploded perspective view illustrating a member
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`configuration of an example of a cell culture chip according to a first exemplary
`
`embodiment;
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`FIG. 1B is a top view of a scaffolding material which is one of members
`
`configuring the cell culture chip:
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`20
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`FIG. 1C is a cross-sectional view of the scaffolding material of FIG. 1B
`
`as viewed in an A-A direction;
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`FIG. 1D is a schematic transmission view illustrating a flow path in the
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`central portion of the cell culture chip in which the scaffolding material of FIG.
`
`1B is incorporated;
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`25
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`FIG. 2 is a flowchart of a method of manufacturing the scaffolding
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`material of the cell culture chip according to the first exemplary embodiment;
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`FIG. 3 is a schematic cross-sectional view illustrating a cross-sectional
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`2
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`

`

`structure of the cell culture chip of FIG. 1D as viewed in a B-B direction;
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`FIG. 4 is a schematic view illustrating a member configuration of an
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`example of a cell culture chip of the related art;
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`FIG. 5 is a schematic cross-sectional view illustrating a cross-sectional
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`structure of a central portion of an example of the cell culture chip of the
`
`related art;
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`FIG. 6A is a schematic transmission view illustrating a planar
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`structure of a flow path of a central portion of an example of a task structure of
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`the cell culture chip of the related art; and
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`10
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`FIG. 6B is a schematic cross-sectional view illustrating a cross-sectional
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`structure as viewed in a D-D direction of FIG. 6A.
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`DETAILED DESCRIPTION
`
`[0006]
`
`FIG. 4 is a schematic view illustrating a memberconfiguration of an
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`15
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`example of a general cell culture chip proposed by Japanese Patent Application
`
`No. 2019-506861. First, members configuring the cell culture chip of the
`
`related art will be described with reference to FIG. 4.
`
`In this cell culture chip,
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`cells are cultured on a sheet of scaffolding material 9’ of the related art formed
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`of a polymer material
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`such as polyethylene phthalate or polystyrene.
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`20
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`Scaffolding material 9’ of the related art is sandwiched betweenfirst partition
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`wall layer 103 and second partition wall layer 104 in which first main flow path
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`3-1 and second main flow path 4-1 for supplying a liquid medium used for cell
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`culture are formed respectively. Widths of first main flow path 3-1 and second
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`main flow path 4-1 are generally formed within a range of 0.2 to 0.5 mm.
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`25
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`[0007]
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`A sandwiching structure between first partition wall layer 103 and
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`second partition wall layer 104 of scaffolding material 9’ is formed by a method
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`of aligning and stacking respective configuration members of first partition
`
`3
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`

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`wall layer 103, scaffolding material 9’, and second partition wall layer 104,
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`interposing an adhesive therebetween, or the like.
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`In addition, first partition
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`wall layer 103 and second partition wall layer 104 are generally formed of
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`silicone resin. The partition wall layers are including first main flow path 3-1
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`and second main flow path 4-1 that serve to be supplied with a medium from
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`the outside of the chip and discharge the medium, and through-holes 5 that
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`serve as alignment marks when first partition wall layer 103 is stacked on
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`second partition wall layer 104.
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`[0008]
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`First board 101 and second board 102 are stacked on external sides
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`10
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`of first partition wall layer 103 and second partition wall layer 104, respectively.
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`First board 101 and second board 102 serve aslids of first main flow path 3-1
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`and second main flow path 4-1 filled with a liquid medium. First board 101
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`and second board 102 are generally formed of glass having a thickness of
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`approximately 0.3 to 1.0 mm, and first partition wall layer 103 and second
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`15
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`partition wall layer 104 are stacked and bonded by a method of interposing an
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`adhesive therebetween or the like.
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`In addition, similar to first partition wall
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`layer 103 or second partition wall layer 104, first board 101 is provided with
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`through-holes
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`2
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`that
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`serve as alignment marks when supplying and
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`discharging a medium from the outside of the chip and stacking respective
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`20
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`configuration members.
`
`[0009]
`
`FIG.
`
`5
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`is a schematic cross-sectional view illustrating a
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`cross-sectional structure of a central portion of cell culture chip 107 after
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`respective configuration members are stacked and bonded. A medium used for
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`cell culture is divided into an upper portion and a lowerportion via scaffolding
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`25
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`material 9’ of the related art in which cells are cultured in first main flow path
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`3-1 of an upper flow path and in second main flow path 4-1 of a lower flow path.
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`[0010]
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`Due to this configuration, for example, when drug is added to the
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`4
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`

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`medium filling first main flow path 3-1 of first partition wall layer 103, the
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`drug reaches a medium filling second main flow path 4-1 of second partition
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`wall layer 104 through scaffolding material 9' of the related art in which the
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`cells are cultured. Thus, by analyzing medium component of second main flow
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`path 4-1 of second partition wall layer 104 after a certain time, it is possible to
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`evaluate drug efficacy, toxicity test or absorption, metabolism, and the like of
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`the drug for the cells. Particularly, as illustrated in FIG. 1 of Japanese Patent
`
`Unexamined Publication No. 2019-180354, when a sheet having a planar mesh
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`structure is used for scaffolding material 9’ of the related art, co-culture (for
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`10
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`example, hepatocytes or intestinal cells, alternatively, cerebral blood barrier
`
`cells, cerebral nerve cells, or the like) of a cell sheet with a function closer to an
`
`organ is possible.
`
`[0011]
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`Further, as a thickness of the sheet having the planar mesh
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`structure is reduced and an aperture ratio is increased, the cell sheets cultured
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`15
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`in first main flow path 3-1 and second main flow path 4-1 are closer to each
`
`other. Therefore, since the function is closer to the organ, it is expected that
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`accuracy of test results of drug efficacy, toxicity test or absorption, metabolism,
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`and thelike of the drug for the cells is increased.
`
`[0012]
`
`However, the general cell culture chip of the related art proposed by
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`20
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`Japanese Patent Application No. 2019-506861 and the like has a problem that
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`evaluation of cells performed by using the cell culture chip is inaccurate.
`
`Specifically, the present disclosure relates to improvement of a cell culture
`
`state and electrical evaluation. FIG. 6A is a schematic transmission view
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`illustrating a planar structure of a flow path of a central portion of cell culture
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`25
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`chip 107 of the related art to which the planar mesh structure sheet having a
`
`small thickness and a large aperture ratio is applied, as scaffolding material 9’
`
`of the related art illustrated in FIG. 5. FIG. 6B is a schematic cross-sectional
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`5
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`

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`view illustrating a cross-sectional structure as viewed in a D-D direction of FIG.
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`6A. When a culture state of cells in the cell culture chip is electrically
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`evaluated by a certain method, a space can be easily formed in cell sheets of
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`upper cells 10-1 and lower cells 10-2, at an end portion where a meshis easily
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`enlarged during culturing, as illustrated in the schematic cross-sectional view
`
`of FIG. 6B. As a result, there is a problem that a short circuit (electrical
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`conduction) occurs.
`
`[0013]
`
`As described above, a problem to be solved by the present disclosure
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`1s to provide a cell culture chip that enables more accurate electrical evaluation
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`10
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`while using a scaffolding material that enables co-culture of cell sheets in the
`
`cell culture chip.
`
`[0014]
`
`A cell culture chip according to a first aspect has a stack structure
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`formed by sequentially stacking a first electrode provided on a main surface of
`
`a first board, a first partition wall layer including a first main flow path, anda
`
`15
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`first inlet flow path anda first outlet flow path connected to the first main flow
`
`path, a planar mesh structure sheet used as a scaffolding material for cells, a
`
`second partition wall layer including a second main flow path, and a second
`
`inlet flow path and a second outlet flow path connected to the second main flow
`
`path, and a second electrode provided on a main surface of a second board, in
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`20
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`which the planar mesh structure sheet
`
`is sandwiched between the first
`
`partition wall layer and the second partition wall layer, among aperture ratios
`
`of a surface of the planar mesh structure sheet facing the first partition wall
`
`layer, the aperture ratio of a portion facing the first main flow path is greater
`
`than the aperture ratios of portions facing thefirst inlet flow path and thefirst
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`25
`
`outlet flow path, and among aperture ratios of a surface of the planar mesh
`
`structure sheet facing the second partition wall layer, the aperture ratio of a
`
`portion facing the second main flow path is greater than the apertureratios of
`
`6
`
`

`

`portions facing the secondinlet flow path and the second outlet flow path.
`
`[0015]
`
`According to a cell culture chip according to a first aspect of the
`
`present disclosure, in s planar mesh structure sheet used as a scaffolding
`
`material, an aperture ratio of a portion facing a first main flow path is greater
`
`than aperture ratios of portions facing a first inlet flow path andafirst outlet
`
`flow path, and an aperture ratio of a portion facing a second main flow path is
`
`greater than aperture ratios of portions facing a second inlet flow path anda
`
`second outlet flow path. Accordingly, it is possible to suppress occurrence of
`
`short circuits (electrical conductions) at a portion wherethefirst inlet flow path
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`10
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`and the second inlet flow path intersect and at a portion from whichthefirst
`
`outlet flow path and the second outlet flow path branch off. Further, it is
`
`possible to provide a state in which cell sheets cultured in the first main flow
`
`path and the second main flow path are closer to each other. Therefore, in the
`
`cell culture chip, it is possible to increase accuracy of evaluation of cells and to
`
`15
`
`perform more stable co-culture (for example, hepatocytes or intestinal cells,
`
`alternatively, cerebral blood barrier cells, cerebral nerve cells, or the like) of a
`
`cell sheet having a function closer to an organ. Thereby, since the function is
`
`closer to the organ, accuracy of test results of drug efficacy, toxicity test or
`
`absorption, metabolism, and the like of drugfor cells is increased.
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`20
`
`[0016]
`
`In a cell culture chip according to a second aspect, in the first aspect,
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`a planar mesh structure sheet used as a scaffolding material is formed by
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`crossing fibers of a polymer material at 30 degrees to 120 degrees, each fiber
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`having a diameter of 1 pm to 50 pm.
`
`[0017]
`
`In a cell culture chip according to a third aspect, in the first aspect or
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`25
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`the second aspect, in the planar mesh structure sheet, an interval between
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`fibers of a mesh structure of a scaffolding material corresponding to a main
`
`portion where an upper flow path and a lower flow path face each other and
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`7
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`

`

`cells are cultured is 10 to 100 pm, and an interval between fibers of a mesh
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`structure of a scaffolding material facing a portion to or from which the upper
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`flow path and the lower flow path mergeor branchoff, is 1 to 10 pm.
`
`[0018]
`
`Hereinafter, a cell culture chip according to an exemplary
`
`embodiment will be described with reference to the accompanying drawings.
`
`In the drawings, substantially the same members are denoted by the same
`
`reference numerals.
`
`First Exemplary Embodiment
`
`[0019]
`
`Hereinafter, cell culture chip 107 according to a first exemplary
`
`10
`
`embodiment will be described.
`
`FIG. 1A is an exploded perspective view
`
`illustrating a member configuration of an example of cell culture chip 107
`
`according to the first exemplary embodiment.
`
`FIG. 1B is a top view of
`
`scaffolding material 9 which is one of members configuring the cell culture chip.
`
`FIG. 1C is a cross-sectional view of the scaffolding material of FIG. 1B as
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`15
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`viewed in an A-A direction.
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`FIG. 1D is a schematic transmission view
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`illustrating a flow path of a central portion of the cell culture chip in which
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`scaffolding material 9 of FIG. 1B isincorporated.
`
`In the drawings, for the sake
`
`of convenience, a direction in which first main flow path 3-1 and second main
`
`flow path 4-1 extend is referred to as an X direction, a width direction
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`20
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`perpendicular to the X direction in a plane is referred to as a Y direction, anda
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`direction perpendicular to an XY planeis referred to as a Z direction.
`
`[0020]
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`Cell culture chip 107 has a stack structure in which first board 101,
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`first partition wall
`
`layer 103, planar mesh structure sheet 9 used as a
`
`scaffolding material, second partition wall layer 104, and second board 102 are
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`25
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`sequentially stacked. First board 1011s provided with first electrode 105 on a
`
`main surface thereof. First partition wall layer 103 includes first main flow
`
`path 3-1, andfirst inlet flow path 3-2 and first outlet flow path 3-3 connected to
`
`8
`
`

`

`first main flow path 3-1. Second partition wall layer 104 includes second main
`
`flow path 4-1 and second inlet flow path 4-2, and second outlet flow path 4-3
`
`connected to second main flow path 4-1. Second board 102 is provided with
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`second electrode 106 on a main surface thereof. Planar mesh structure sheet 9
`
`is sandwiched betweenfirst partition wall layer 103 and second partition wall
`
`layer 104. Among aperture ratios of a surface of planar mesh structure sheet
`
`9 facing first partition wall layer 103, the aperture ratio of a portion facing first
`
`main flow path 3-1 is greater than the aperture ratios of portions facing first
`
`inlet flow path 3-2 andfirst outlet flow path 3-3. Among aperture ratios of a
`
`10
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`surface of planar mesh structure sheet 9 facing second partition wall layer 104,
`
`the aperture ratio of a portion facing second main flow path 4-1 is greater than
`
`the aperture ratios of portions facing second inlet flow path 4-2 and second
`
`outlet flow path 4-3. Accordingly, it is possible to suppress occurrence of short
`
`circuits (electrical conductions) at a portion wherefirst inlet flow path 3-2 and
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`15
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`secondinlet flow path 4-2 intersect and at a portion from whichfirst outlet flow
`
`path 3-3 and second outlet flow path 4-3 branch off. Further, it is possible to
`
`provide a state in which thecell sheets cultured in first main flow path 3-1 and
`
`second main flow path 4-1 are closer to each other. Therefore, in the cell
`
`culture chip, it is possible to increase accuracy of evaluation of cells and to
`
`20
`
`perform more stable co-culture (for example, hepatocytes or intestinal cells,
`
`alternatively, cerebral blood barrier cells, cerebral nerve cells, or the like) of a
`
`cell sheet having a function closer to an organ. Thereby, since the function is
`
`closer to the organ, accuracy of test results of drug efficacy, toxicity test or
`
`absorption, metabolism, and the like of drugforcells is increased.
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`25
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`[0021]
`
`Hereinafter, respective members configuring cell culture chip 107
`
`will be described in detail. Cell culture chip 107 is formed by sequentially
`
`aligning, stacking, and bonding respective configuration membersof first board
`
`9
`
`

`

`101, first partition wall layer 103 having first main flow path 3-1, planar mesh
`
`structure sheet 9 used as a scaffolding material of cells, second partition wall
`
`layer 104 having second main flow path 4-1, and second board 102.
`
`First Board and Second Board
`
`[0022]
`
`On a side of first board 101 facing first partition wall layer 103, first
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`electrode 105 is formed of a wire, which has a width smaller than or equal to a
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`width of first main flow path 3-1, along an internal side of first main flow path
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`3-1 and a wide wire drawn from the wire. Likewise, on a side of second board
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`102 facing second partition wall layer 104, second electrode 106 is formedof a
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`10
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`wire, which has a width smaller than or equal to a width of second main flow
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`path 4-1, along an internal side of second main flow path 4-1 and a wide wire
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`drawn from the wire. By using first electrode 105 and second electrode 106,
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`electrical resistance can be increased by forming a tight junction or the lke
`
`between a cell sheet of upper cells 10-1 (ot illustrated) to be cultured in first
`
`15
`
`main flow path 3-1 and a cell sheet of lower cells 10-2 (not illustrated) to be
`
`cultured in second main flow path 4-1. As a result, culture states of the cell
`
`sheets can be stably evaluated.
`
`[0023]
`
`Glass may be usedfor first board 101 and second board 102. Both
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`first electrode 105 and second electrode 106 may be formed of indium tin oxide
`
`20
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`(ITO). Accordingly, since first board 101 and second board 102 and first
`
`electrode 105 and second electrode 106 are all transparent, cells to be cultured
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`can be visually evaluated by using a microscope or the like.
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`In addition to
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`glass, a resin material such as polystyrene or acrylic may be used for first board
`
`101 and second board 102, but it is preferable that first board 101 and second
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`25
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`board 102 are transparent from the above viewpoint.
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`First Partition Wall Layer and Second Partition Wall Layer
`
`[0024]
`
`First partition wall layer 103 includes first main flow path 3-1, and
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`10
`
`

`

`first inlet flow path 3-2 and first outlet flow path 3-3 connected to first main
`
`flow path 3-1. Second partition wall layer 104 includes second main flow path
`
`4-1 and second inlet flow path 4-2, and second outlet flow path 4-3 connected to
`
`second main flow path 4-1.
`
`In the present exemplary embodiment,
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`polystyrene resin may be used for a molding material for forming first partition
`
`wall layer 103 having first main flow path 3-1 and second partition wall layer
`
`104 having second main flow path 4-1. Adhesion of first board 101 and first
`
`partition wall layer 103 and adhesion of second board 102 and second partition
`
`wall layer 104 can be made by leaving the boards and partition wall layers in
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`10
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`an atmosphere of 80°C for approximately 2 hours after adhesive 6 is
`
`dispense-coated and the boards and partition wall layers are aligned and
`
`stacked. Alternatively, thermocompression bonding may be used as a method
`
`that does not use an adhesive. Here, when an adhesiveis used,it is preferable
`
`to use a silicone-based material from a viewpoint of cytotoxicity.
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`15
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`Planar Mesh Structure Sheet (Scaffolding Material)
`
`[0025]
`
`Planar meshstructure sheet 9 used as a scaffolding material for cells
`
`is sandwiched betweenfirst partition wall layer 103 and second partition wall
`
`layer 104 in which first main flow path 3-1 formed infirst partition wall layer
`
`103 and second main flow path 4-1 formed in second partition wall layer 104
`
`20
`
`face each other and which are stacked so as to overlap each other. A method of
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`sandwiching planar mesh structure sheet 9 used as a scaffolding material can
`
`be performed by dispense-coating first partition wall layer 103 and second
`
`partition wall layer 104 with an adhesive and aligning and stacking the
`
`partition wall
`
`layers, and then leaving the partition wall
`
`layers in an
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`25
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`atmosphere
`
`of
`
`80°C for
`
`approximately
`
`2
`
`hours.
`
`Alternatively,
`
`thermocompression bonding may be used as a method that does not use an
`
`adhesive.
`
`11
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`

`

`[0026]
`
`Planar mesh structure sheet 9 used as a scaffolding material is, for
`
`example, a planar mesh structure sheet formed of a fiber group of polystyrene.
`
`Further, planar mesh structure sheet 9 has, for example, a thickness of
`
`approximately 2 pm, an interfiber distance of approximately 3 pm in an outer
`
`circumferential portion thereof, and an interfiber distance of approximately 20
`
`im in a central portion thereof but is not limited thereto. Since a thickness
`
`corresponds to a diameter of a fiber,
`
`the thickness may be appropriately
`
`adjusted according to characteristics of the type of cells to be cultured but is
`
`preferably 1 pm to 50 pm. Further, interfiber distances corresponding to a
`
`10
`
`portion wherefirst inlet flow path 3-2 and second inlet flow path 4-2 intersect
`
`and a portion from which first outlet flow path 3-3 and second outlet flow path
`
`4-3 branch off is preferably less than or equal to 10 ym from a viewpoint of
`
`suppressing occurrence of a short circuit (electrical conduction) even in a
`
`portion where uppercells 10-1 and lowercells 10-2 are difficult to be cultured in
`
`15
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`a sheet shape. Furthermore, cell sheets cultured in first main flow path 3-1
`
`and second main flow path 4-1 may be closer to each other but an interfiber
`
`distance corresponding to first main flow path 3-1 and second main flow path
`
`4-1 is preferably greater than or equal to 10 pm orless than or equal to 100 pm
`
`whenconsidering an initial cell size to be cultured. Further, polystyrene with
`
`20
`
`less concern about cytotoxicity described above can be used for resin forming
`
`scaffolding material 9, but the resin is not limited thereto as long as concern
`
`about cytotoxicity is less. Resin for forming scaffolding material 9 may be, for
`
`example, a polylactic acid type or a silicone type, but is preferably a polymer
`
`material becauseflexibility is required as a function as a scaffold of cells.
`
`Ina
`
`25
`
`cross-sectional view of planar mesh structure sheet 9 of FIG. 1C as viewed in an
`
`A-A direction, planar mesh structure sheet 9 which is a scaffolding material
`
`has a planar mesh structure configured by a two-layer structure of first layer
`
`12
`
`

`

`spinning group 108 and second layer spinning group 109. First layer spinning
`
`group 108 and second layer spinning group 109 are bonded by being partially
`
`entwined, which will be described below.
`
`[0027]
`
`Furthermore, a sparse and dense structure that is characteristics of
`
`planar mesh structure sheet 9 will be described in detail with reference to a
`
`schematic transmission view illustrating a flow path of a central portion of the
`
`cell culture chip of FIG. 1D. As illustrated in FIG. 1D, a portion wherefibers
`
`of planar mesh structure sheet 9 which is the scaffolding material of FIG. 1C is
`
`dense, that is, a portion of a mesh with a small aperture ratio faces a portion to
`
`10
`
`whichfirst inlet flow path 3-2 and second inlet flow path 4-2 merge or a portion
`
`from whichfirst outlet flow path 3-3 and secondoutlet flow path 4-3 branch off.
`
`For example, the portion of the mesh with the small aperture ratio is alternate
`
`long and short dash hne B-Bin FIG. 1D. Further, a portion where fibers are
`
`sparse, that is, a portion of a mesh with a large aperture ratio faces a main
`
`15
`
`portion where cells are cultured in first main flow path 3-1 and second main
`
`flow path 4-1, for example, a C region in FIG. 1D.
`
`Method of Manufacturing Planar Mesh Structure Sheet 9 which is Scaffolding
`
`Material
`
`[0028]
`
`FIG. 2 is a flowchart of a method of manufacturing planar mesh
`
`20
`
`structure sheet 9 which is a scaffolding material for forming the cell culture
`
`chip according to the first exemplary embodiment.
`
`[0029]
`
`(1) SO1 is a process of preparing a film.
`
`It is preferable that a film
`
`surface has appropriate peeling off in fluorine processing or the like. This is
`
`because an adhesive function to a fiber is required when spinningthefiber on a
`
`25
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`film in S02 and S04 to be described below, and in the first exemplary
`
`embodiment,
`
`the peeling off function from the film is required when
`
`incorporating scaffolding material 9 into the cell culture chip later.
`
`13
`
`

`

`[0030]
`
`(2) S02 is a process of spinning a first layer. A solution obtained by
`
`melting a polymer material used as scaffolding material 9 for the cell culture
`
`chip according to the first exemplary embodiment by heating or swelling with
`
`an organic solvent is applied onto a film prepared in S01 in a fine line shape in
`
`the same direction. Here,
`
`the polymer material supplied in a melting or
`
`solution state is naturally cooled or naturally dried to form a fiber only in a
`
`solid state.
`
`In the first exemplary embodiment, for example, polystyrene with
`
`low cytotoxicity is employed as the polymer material, and fibers, each having a
`
`diameter of approximately 2 pm, are coated, in the same direction at equal
`
`10
`
`intervals of 3 pm, with a solution obtained by swelling pellet-shaped
`
`polystyrene in N,N-dimethylformamide (N-DMF) which is an organic solvent
`
`by 30% by weight.
`
`[0031]
`
`(3) S03 is a process of rotating the film, which is obtained by
`
`spinning thefirst layer in S02, at a predetermined angle in the same surface.
`
`15
`
`In the first exemplary embodiment, a mesh structure is formed in which the
`
`first layer spun in SO1 and a second layer to be spun in S04 to be described
`
`below are orthogonal to each other by being rotated by 90 degrees. However, a
`
`crossing angle maybe 30 degrees to 120 degrees, but it is preferable to cross at
`
`90 degrees from a viewpoint of holding a meshof a scaffolding material against
`
`20
`
`an external force.
`
`[0032]
`
`(4) S04 is a process of spinning the second layer on thefilm rotated
`
`by 90 degrees in S038. A solution obtained by melting a polymer material used
`
`as scaffolding material 9 by heating or swelling with an organic solvent is
`
`applied onto a film prepared in S03 in a fine line shape in the same direction.
`
`25
`
`In the first exemplary embodiment, polystyrene with low cytotoxicity is
`
`employed as the polymer material in the same manneras in S02, and fibers,
`
`each having a diameter of approximately 2 ym, are coated,
`
`in the same
`
`14
`
`

`

`direction, with a solution obtained by swelling pellet-shaped polystyrene in
`
`N,N-dimethylformamide (N-DMF) which is an organic solvent by 30% by
`
`weight. However, as illustrated in the cross-sectional view of FIG. 1C, a first
`
`half of the coating is at an interval of 3 ym, a middle stage is at an interval of
`
`20 pm, and a second half is at an interval of 3 pm.
`
`[0033]
`
`(5) S05 is a process of heating the fiber mesh on the film prepared up
`
`to process S04. Specifically, by heating the polymer material (polystyrene in
`
`the first exemplary embodiment) at a temperature higher than or equal to a
`
`glass transition point and lower than a melting point for a certain time, a
`
`10
`
`majority portion of contact points between an upper portion of the first layer
`
`and a lower portion of the second layer is entwined. A planar mesh structure
`
`sheet used as a scaffolding material can be obtained based on the above.
`
`[0034]
`
`Accordingly,
`
`a scaffolding material, which faces an outer
`
`circumferential portion of the cell culture chip, that is, a portion to which first
`
`15
`
`inlet flow path 3-2 and second inlet flow path 4-2 merge or a portion from which
`
`first outlet flow path 3-3 and second outlet flow path 4-3 branchoff, has a dense
`
`meshstructure and a small mesh opening, that is, an aperture ratio of a mesh
`
`is reduced. Meanwhile, a scaffolding material, which faces a central portion of
`
`the cell culture chip, that is, a main portion where cells are cultured in first
`
`20
`
`main flow path 3-1 and second main flow path 4-1, has a sparse mesh structure
`
`and a large mesh opening, that is, an aperture ratio of a meshis increased.
`
`[0035]
`
`FIG.
`
`3
`
`is
`
`a
`
`schematic cross-sectional view illustrating a
`
`cross-sectional structure of the cell culture chip of FIG. 1D as viewed in a B-B
`
`direction.
`
`In FIG. 3, in a region D of an outer circumferential portion of the
`
`25
`
`cell culture chip, a mesh is easily enlarged during culturing at a portion from
`
`which first outlet flow path 3-3 and second outlet flow path 4-3 branch off.
`
`Therefore, a space can be easily formed in cell sheets of upper cells 10-1 and
`
`15
`
`

`

`lower cells 10-2, and cells are difficult
`
`to be cultured in a sheet shape.
`
`However, in the cell culture chip according to the first exemplary embodiment,
`
`by using scaffolding material 9, a portion having a dense mesh structure and a
`
`small mesh opening, that is, a portion having a small aperture ratio of a mesh
`
`faces the portion from which first outlet flow path 3-3 and second outlet flow
`
`path 4-3 branch off. Therefore, even when a meshis shghtly large during
`
`culturing, formation of a gap is prevented and a scaffolding material itself
`
`becomeselectrical resistance, and thus, it is possible to suppress occurrence of a
`
`short circuit (electrical conduction). The same applies to a portion to which
`
`10
`
`first inlet flow path 3-2 and second inlet flow path 4-2 merge.
`
`[0036]
`
`Thereby, electrical resistance can be increased by forming a tight
`
`junction or the like between a cell sheet of upper cells 10-1 to be cultured in
`
`first main flow path 3-1 and a cell sheet of lower cells 10-2 to be cultured in
`
`second main flow path 4-1. Asa result, it is possible to suppress occurrence of
`
`15
`
`a short circuit, and to stably evaluate a culture state of a cell sheet.
`
`[0037]
`
`Further, in the first exemplary embodiment, in order to make an
`
`aperture ratio of a planar mesh structure sheet greater in a first main flow path
`
`than in a first inlet flow path andafirst outlet flow path and to make an
`
`aperture ratio of a planar mesh structure sheet greater in a second main flow
`
`20
`
`path than in a secondinlet flow path and a second outlet flow path, an interval
`
`between fibers is controlled by keeping a fiber diameter constant, but the
`
`present disclosure is not limited to this method. For example, a fiber diameter
`
`may be controlled by keeping an interval between fibers constant.
`
`[0038]
`
`Specifically, for example, a fiber diameter of second layer spinning
`
`25
`
`group 109 is set to 8 pm in a first half, 2 pm in a middle stage, and 8 pm ina
`
`second half. Accordingly, a scaffolding material, which faces a portion to
`
`whichfirst inlet flow path 3-2 and second inlet flow path 4-2 merge, or a portion
`
`16
`
`

`

`from which first outlet flow path 3-3 and second outlet flow path 4-3 branch off,
`
`has a dense mesh structure and a small mesh opening, that is, an aperture
`
`ratio of a mesh can be reduced. Meanwhile, a scaffolding material, which
`
`faces a main portion where cells are cultured in first main flow path 3-1 and
`
`second main flow path 4-1, has a sparse mesh structure and a large mesh
`
`opening, that is, an aperture ratio of a mesh can be increased.
`
`[0039]
`
`The present disclosure includes appropriate combination of any
`
`
`
`
`
`
`
`exemplary among_variousembodiment and/or example exemplary
`
`
`
`
`
`embodiments and/or examples described above, and effects of the respective
`
`10
`
`exemplary embodiments and/or examples can be obtained.
`
`[0040]
`
`According to a cell culture chip of the present disclosure, in thecel