* NOTICE *
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`JPO and INPIT are not responsible for any damages caused by the use of this translation.
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`1. This document has been translated by computer. So the translation may not reflect the original precisely.
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`2. **** shows a word which cannot be translated.
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`3. in the drawings, any words are not translated.
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`Publication Number
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`JP2020010641A
`
`Bibliography
`
`(19) [Publication country] JP
`
`(12) [Kind of official gazette] A
`
`(11) [Publication number] 2020010641
`
`(43) [Date of publication of application] 20200123
`
`(54) [Title of the invention] CELL CULTURE APPARATUS AND CELL SEEDING
`
`METHOD
`
`(51) [International Patent Classification]
`
`C12M 3/06
`
`C12M 1/12
`
`C12M 1/26
`
`(2006.01)
`
`(2006.01)
`
`(2006.01)
`
`
`
`C12N=1/02 (2006.01)
`
`BOD 63/00
`
`(2006.01)
`
`[Fi]
`
`C12M 3/06
`
`C12M—so1/12
`
`C12M=1/26
`
`C12N=1/02
`
`BO1D 63/00
`
`500
`
`(21) [Application number] 2018135307
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`(22) [Filing date] 20180718
`
`(71) [Applicant]
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`[Name] SHIMADZU CORP
`
`[Name] TOKYO INSTITUTE OF TECHNOLOGY
`
`(72) [Inventor]
`
`[Full name] MIYAKE TSUTOMU
`
`

`

`[Full name] FUJIYAMA YOICHI
`
`[Full name] TAGAWAYOICHI
`
`[Theme code (reference)}
`
`4B029
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`4B065
`
`4D006
`
`[F-term (reference) ]
`
`4B029AA08
`
`4B029AA09
`
`4B029AA27
`
`4B029BB11
`
`4B029CC01
`
`4B029DG08
`
`4B029GB05
`
`4B029HA0S
`
`4B065BD18
`
`4D006GA02
`
`4DO006HA41
`
`4DO06JA05A
`
`4D006JA05C
`
`4ADO006JA06A
`
`4D006JA06C
`
`4D006JA14A
`
`ADO06JA18A
`
`4D006JA23A
`
`4D006JA23C
`
`4D006JA53Z
`
`4D006KE22Q
`
`4ADO06MAO3
`
`4DO06MA18
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`4D006PB20
`
`ADO06PB70
`
`Abstract
`
`(57) [Overview]
`
`PROBLEM TO BE SOLVED: To provide a ceil culture apparatus capable of inhibiting
`
`

`

`cell culture (analysis) from becoming inappropriate because an environment modeling
`
`the inside of a living body is broken due to cells not being properly seeded on a
`
`permeable membrane.
`
`SOLUTION: A cell culture apparatus 100 comprises: a permeable membrane 1; flow
`
`passage members 2 disposed above the permeable membrane 1 and including flow
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`passage members 20, 21; and flow passage members 4 disposed below the permeable
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`membrane 1 and including flow passage members 40, 41. The flow passage member 20
`
`has an introduction port 20a and a drain port 20b. The flow passage member40 has a
`
`drain port 406 and an introduction port 40a. The cell culture apparatus 100 is configured
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`such that a cell suspension is introduced through the introduction port 20a, cells 101
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`contained in the cell suspension are seeded on the permeable membrane 1, and liquid
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`medium in the cell suspension passed through the permeable membrane 1 is drained
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`through the drain port 40b.
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`SELECTED DRAWING: Figure 1
`
`Claim
`
`{Patent Claims]
`
`[Claim 1]
`
`A permeable membrane permeabie to the liquid medium without permeating the cell
`
`against a cell suspension suspended in a liquid medium ;
`
`A one side channel member which is arranged on one side of the permeable membrane
`
`and includes one side 1 channel member which has one side introduction part and one
`
`side discharge part, and a one side 1 channel member whichis arranged between the
`
`one side 2 channel member and the transmission membrane and has one side channel
`
`which is electrically connected to the one side introduction part and the one side
`
`discharge part ;
`
`A 1
`
`flow passage memberis provided which is disposed on the other side of the
`
`permeable membrane and has the other-side second flow passage memberhaving the
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`other-side discharge portion and the other-side introduction portion, and the other-side
`
`2 flow passage memberthat is disposed between the other-side 1 flow passage member
`
`and the transmissive film and has the other-side flow passage that is electrically
`
`connected to the other-side introduction portion and the other-side discharge portion.
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`The ceil culture apparatus is configured so that, by introducing the cell suspension from
`
`the one side introduction section, the cells contained in the ceil suspension are seeded
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`on the permeable membrane and the liquid medium contained in the cell suspension
`
`permeated through the permeable membrane is discharged from the other-side
`
`

`

`discharge section.
`
`[Claim 2]
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`The one side flow channel memberis arranged on an upper side of the permeable
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`membrane.
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`The cell culture apparatus according to claim 1, wherein the other-side channel member
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`is disposed on a lowerside of the permeable membrane.
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`[Claim 3]
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`Each of the side of the one side introduction part and the side of the other side discharge
`
`part is opened and opened. The ceil culture apparatus according to claim 1 or 2, wherein,
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`by closing each of the one side outlet and the other-side inlet, the cell suspension is
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`introduced from the one side inlet and the liquid medium is discharged from the other-
`
`side outlet.
`
`[Claim 4]
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`A valve for one side introduction part which is arranged upstream of the one side
`
`introduction part and controls conduction of the cell suspension to the one side
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`introduction part ;
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`A valve for one side discharge part arranged downstream of the one side discharge part ;
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`A valve for the other side introduction part arranged upstream of the other-side
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`introduction part ;
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`Further, a valve for the other side discharge part which is arranged downstream of the
`
`other-side discharge part and controls conduction of the liquid medium from the other-
`
`side discharge part is further provided.
`
`Both the valve for the one side introduction part and the valve for the other side discharge
`
`part are opened and the valve is opened. The cell culture apparatus according to any
`
`one of claims 1
`
`to 3, wherein the cell suspension is introduced from the one side
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`introduction portion and the liquid medium is discharged from the other-side discharge
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`portion in a state in which both of the one side discharge portion vaive and the other-side
`
`introduction portion valve are closed.
`
`[Claim 5]
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`A pump for one side introduction part is arranged upstream of the one side introduction
`
`part.
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`The cell culture apparatus according to any one of claims 1
`
`to 4, wherein the cell
`
`suspension is introduced from the one side introduction portion and the liquid medium is
`
`discharged from the other-side discharge portion by driving the one side introduction
`
`portion pump.
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`[Claim 6]
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`

`

`A 1 seal member which is laminated on one side of the permeable membrane and
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`includes a one side opening part which exposes a predetermined area of the permeabie
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`membrane;
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`A 2 seal member which is laminated on the other side of the permeable membrane and
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`includes the second side opening portion which exposes a region corresponding to the
`
`predetermined region of the transmissive membrane is provided.
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`The cell suspension introduced from the one side introduction part is conducted to the
`
`one side opening part of the 1 seal member, and the cells are seeded in the
`
`predetermined area of the permeation membrane.
`
`The liquid medium passing through the predetermined area of the permeable membrane
`
`is conducted from the other side opening of the 2 seal member to the other side
`
`discharge part, and is discharged from the other side discharge part.
`
`A cell culture apparatus as claimed in any one of claims 1 to 5, wherein said ceil culture
`
`apparatus comprises a cell culture apparatus.
`
`[Claim 7]
`
`The other side discharge part has a 1 other-side discharge port and a 1 other-side
`
`discharge port different from the 2 other-side discharge port.
`
`The predetermined region includes a 1 region and a 1 region different from the 2 region.
`
`The other side opening of the 2 seal member has a 1 side opening part exposing the 1
`
`area of the transmission film and a 2 side opening part exposing the 2 area of the
`
`transmission film.
`
`The cell comprises a 1 cell and a 2 cell.
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`By conducting the cell suspension from the one side introduction part to the one side
`
`opening part of the 1 seal member, the 1 cell is seeded in the 1 area of the permeable
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`membrane, and the liquid medium transmitted through the 1 area of the permeable
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`membrane is transmitted. The 1 side opening portion is electrically connected to the 1
`
`side discharging port, and the 1 side opening portion is connected to the second side
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`opening portion. The cell culture apparatus according to claim 6, wherein the 2 ceil is
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`seeded in the 2 region of the permeable membrane, and the liquid medium that has
`
`passed through the 2 region of the permeable membrane is conducted from the 2 side
`
`opening portion to the 2 side discharge port and is discharged from the 2 side discharge
`
`port.
`
`[Claim 8]
`
`The 1 side flow passage memberhas the 1 side discharge port and the 2 side discharge
`
`port.
`
`

`

`The cell culture apparatus according to claim 7, wherein the 2 side flow path member
`
`has a 1 side flow path connecting the 1 side opening peortion and the 1 side discharge
`
`port, and a 2 side flow path connecting the 2 side opening portion and the 2 side
`
`discharge port and separated from the 1 side flow passage.
`
`[Claim 9]
`
`A step of introducing the cell suspension from one side introduction part of one side
`
`introduction part and one side ejection part arranged on one side of a permeable
`
`membrane permeating the liquid medium without permeating the cell against a cell
`
`suspension suspendedin a liquid medium ;
`
`A step of circulating the cell suspension introduced from the one side introduction part to
`
`the permeable membrane via a one side flow passage arranged between the one side
`
`introduction part and the one side discharge part and the permeable membrane and
`
`communicating with the one side introduction part and the one side discharge part ;
`
`A step of seeding the cells contained in the ceil suspension circulated through the one
`
`side channel with the permeable membrane and transmitting the liquid medium
`
`contained in the cell suspension to the other side of the permeable membrane;
`
`A step of circulating the liquid medium that has passed through the permeabie
`
`membrane through the other side channel disposed on the other side of the permeable
`
`membrane;
`
`A cell seeding method comprising a step of discharging the liquid medium flowing
`
`through the other side channel from the other side discharge section of the other side
`
`channel and the other side of the other side channel which is arranged on the opposite
`
`side of the transmission membrane with respect to the other-side channel and whichis
`
`electrically connected to the other-side channel.
`
`Description
`
`[Detailed description of the invention]
`
`[Technical field]
`
`[0001]
`
`The present invention relates to a cell culture apparatus and a cell seeding method, and
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`more particularly, to a cell culture apparatus and a cell seeding method for seeding a
`
`permeable membrane with cells.
`
`[Background of the Invention]
`
`[0002]
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`Conventionally, a cell culture apparatus and a cell seeding method for seeding cells on
`
`a permeable membrane have been known (see, for example, Non-Patent Document 1).
`
`

`

`{0003}
`
`The ceil culture apparatus of Non-Patent Document 1 includes an inlet for introducing a
`
`medium and a cell suspension into a culture container, and an outlet for discharging a
`
`medium in the culture container. Introduction Port and Discharge Port
`
`it is arranged on one side of a membrane for seeding cells (intestinal epithelial cells)
`
`(membrane for seeding cells).
`
`{0004}
`
`in culturing cells, a mucin layer is formed on the membrane which prevents entry of
`
`bacteria or the like. After completion of seeding (and formation of the cell membrane)
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`and formation of the mucin layer, a medium (not containing oxygen) is passed through
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`the membrane (a channel connecting the inlet and the outlet). At the same time, a
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`medium (containing oxygen) is circulated through a channel provided on the other side
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`of the membrane. Thus, the cell culture apparatus can be configured to simulate an in-
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`vivo (intestinal tract).
`
`fOOO5]
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`Also, cells suspended in celf suspensions tend to precipitate downward because of their
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`high specific gravity relative to the liquid medium. In the cell culture apparatus of Non-
`
`Patent Document 1, cells are seeded on a membrane by precipitating celis in a cell
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`suspension between an inlet and an outlet arranged on one side of a membrane (one
`
`channel). Specifically, when a predetermined time has elapsed in a state in which a cell
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`suspension which is stationary between an inlet and an outlet (a channel on one side) is
`
`filled, cells in the ceil suspension precipitate downward.
`
`{Prior art reference]
`
`[Non-patent documents]
`
`[0008]
`
`[Non-patent document 1]Pranjul Shah, et al., “A microfluidics-based in vitro model of the
`
`gastrointestinal human-microbe interface" Nature Communications. 2016;7:41535.
`
`{Summary of the invention]
`
`[Problem to be solved by the invention]
`
`[0007]
`
`However, in the cell culture apparatus described in Non-Patent Document 1, a method
`
`is used in which cells contained in a cell suspension in a channel are precipitated and
`
`seeded on a membrane. Thus, sedimentation of celis may be accumulated in a portion
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`other than a membrane of a cell suspension. In particular, in a (U-shaped) channel in
`
`which both the inlet and the outlet are arranged on one side with respect to the
`
`

`

`membrane, since the channel extends downward from each of the inlet and the outlet,
`
`cells contained in the cell suspension in the channel below the inlet and the outlet may
`
`precipitate and accumulate directly under the inlet and the outlet. Since the medium is
`
`suitably supplied to the cells seeded on the membrane via the membrane during cell
`
`culture, there is a case where the culture medium is difficult to be supplied to the cells
`
`that are precipitated in portions other than the membrane. In addition, when precipitation
`
`is accumulated, it may be difficult to supply a medium to the cells buried in the bottom of
`
`the precipitate or the like. In these cases, there is a disadvantage that the culture medium
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`is not properly supplied to the cells that are precipitated and accumulated in portions
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`other than the membrane, and the cells die. If dead cells are present, the dead celis may
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`adversely affect cells seeded on the membrane. In this case, there is a problem in that
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`the environment in the cell culture apparatus imitating the inside of the living body
`
`collapses and # becomesdifficult to appropriately culture (analyze) the cells.
`
`{0008}
`
`it is made in order to solve the above problems, and this invention is one object of this
`
`invention,
`
`To provide a cell culture device and a ceil seeding method capable of
`
`suppressing inappropriate ceil culture (analysis) of cells due to collapse of an
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`environment simulating a living body in which cells are not properly seeded on a
`
`permeabie membrane.
`
`{Meansfor solving the problem]
`
`{0009}
`
`To achieve the above object, a cell culture apparatus according to the 1 aspect of the
`
`present invention is provided. A permeable membrane which transmits a liquid medium
`
`without permeating cells against a cell suspension suspended in a liquid medium ; a 1
`
`channel member which is arranged on one side of the permeable membrane and has
`
`one side introduction part and one side discharge part ; and one side introduction part
`
`and a 1 permeation part;
`
`One side channel memberincluding a one side 2 channel member which is connected
`
`to the side discharge unit and has one side channel ; and a 1 side channel memberwhich
`
`iS arranged at the other side of the transmission membrane and has the other-side side
`
`discharge unit and the other-side side introduction unit ; A 1 flow passage memberis
`
`provided between the second flow passage member on the other side and the 2 flow
`
`passage memberon the other side having the other side flow passage which is arranged
`
`between the second side introduction portion and the second side discharge portion and
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`which is electrically connected to the other side introduction portion, and the ceil
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`

`

`suspension is introduced from the one side introduction portion. The cell suspension
`
`contained in the cell suspension is seeded on the permeable membrane, and the liquid
`
`medium contained in the cell suspension permeated through the permeable membrane
`
`is discharged from the other side discharge part.
`
`{0010]
`
`in the cell culture apparatus according to the 1 aspect of the present invention, as
`
`described above,
`
`in a state in which cells are seeded on a permeable membrane, a
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`culture solution is allowed to flow through each of the one side channel and the other
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`side channel, whereby the cell culture apparatus can be configured to simulate a living
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`body. in addition, cell suspensions can be seeded byfiltering them through a permeable
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`membrane, with the cell suspension being introduced from one side introduction section
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`and the liquid medium being discharged from the other side discharge section, with the
`
`cell suspension flowing at a constant flow rate. As a result, it is possible to suppress
`
`accumulation of cells in portions other than the permeable membrane, unlike when cells
`
`in a stationary cell suspension are precipitated and seeded on a permeable membrane.
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`As a result, it is possible to reduce the dead cells due to the fact that the medium is not
`
`supplied through the permeable membrane. As a result, it is possible to prevent the dead
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`cells from adversely affecting the cells seeded on the permeable membrane, and to
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`prevent the environment in the cell culture apparatus simulating the living body from
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`being destroyed by the cells affected by the adverse effect. As a result, the environment
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`simulating the living body collapses due to the fact that cells are not properly seeded on
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`the permeable membrane, so that it is possible to suppress inappropriate cell culture
`
`(analysis).
`
`In addition, seeding of ceils means trapping of celis into a permeable
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`membrane.
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`{001 14]
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`In the cell culture apparatus according to the 1 aspect, preferably, the one side channel
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`member is disposed on an upper side of the permeable membrane, and the other side
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`channel memberis disposed on a lower side with respect to the permeable membrane.
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`With this configuration, since the cell suspension (liquid medium) is subjected to
`
`downward force due to its own weight,
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`it is possible to relatively easily flow the cell
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`suspension from the one side channel member(one side introduction portion) side on
`
`the upper side to the lower permeation membrane. Further, the liquid medium that has
`
`passed through the permeable membrane can be relatively easily flowed from the upper
`
`permeable membrane toward the lower side channel! member(the other side discharge
`
`portion).
`
`[0012]
`
`

`

`in the cell culture device in the 1st aspect of affairs of the above -- preferable Each of
`
`the side of the one side introduction portion and the side of the other side discharge
`
`portion is opened, and each of the side of the one side discharge portion and the side of
`
`the other side introduction portion is closed, whereby the cell suspension is introduced
`
`from the oneside introduction portion and the liquid medium is discharged from the other-
`
`side discharge portion. With this configuration, by closing each of the side ofthefirst side
`
`and the side of the second side,
`
`it
`
`is possible to suppress movement of the cell
`
`suspension (liquid medium) introduced from the first side toward the first side and the
`
`second side of the second side. As a resull, it is possible to more efficiently flow the liquid
`
`medium toward the other side discharge portion.
`
`[0013]
`
`in the cell culture apparatus in the 1 aspect, preferably, the one side introduction section
`
`valve for the one side introduction section, the one side introduction section valve for
`
`controlling the conduction to the one side introduction section of the cell suspension, and
`
`the other side introduction section valve arranged at the upstream side of the other-side
`
`introduction section are arranged. Further, a valve for the other side discharge part which
`
`is arranged downstream of the other side discharge part and controls conduction of the
`
`liquid medium from the other side discharge part, and a vaive for the other side discharge
`
`part are both opened and both of the vaive for the one side introduction part and the
`
`valve for the other side discharge part are opened. In a state in which both of the valve
`
`for one side and the valve for the other side are closed, a cell suspension is introduced
`
`from one side introduction part and a liquid medium is discharged from the other side
`
`discharge part. In this configuration, both the valve for the one side introduction section
`
`and the valve for the other side discharge section are opened and the otherside is
`
`opened. By closing both the one side outlet valve and the other side inlet valve, a cell
`
`suspension (liquid medium) introduced from one side inlet portion can be easily flowed
`
`to the other side outlet portion side provided with the other side outlet valve.
`
`[0014]
`
`in the cell culture apparatus in the 1 aspect, preferably, the cell culture apparatus further
`
`comprises a pump for one side introduction section disposed upstream of the one side
`
`introduction section, and is configured so that, by driving the oneside introduction section
`
`pump, the cell suspension is introduced from the one side introduction section and the
`
`liquid medium is discharged from the other-side ejection section. According to this
`
`configuration, by using the one side introduction portion pump, the cell suspension
`
`introduced from the one side introduction portion can be easily flowed at a predetermined
`
`

`

`flow rate (flow rate) or more. As a result, even when the cells accumulate in portions
`
`other than the permeable membrane, the cells accumulated in the portions other than
`
`the permeable membrane can be easily transported to the permeable membrane side
`
`by the pressure of the cell suspension having a predetermined flow rate (flow rate) or
`
`more. As a resuit, it is possible to effectively suppress the accumulation of the cells in
`
`portions other than the permeable membrane.
`
`[0015]
`
`in the cell culture apparatus in the 1 aspect, preferably, the 1 seal memberis laminated
`
`on one side of the permeable membrane andincludesa first seal memberthat exposes
`
`a predetermined area of the permeable membrane, and a 2 seal member that is
`
`laminated on the other side of the permeable membrane and includes the other side
`
`opening that exposes a region corresponding to a predetermined region of the
`
`permeable membrane. Further,
`
`the ceil suspension introduced from the one side
`
`introduction part is conducted to the one side opening part of the 1 seal member. A cell
`
`is seeded in a predetermined region of a permeable membrane, and a liquid medium
`
`passing through a predetermined region of the permeable membraneis conducted from
`
`the other side opening of the 2 seal memberto the other side discharge portion and is
`
`discharged from the other side discharge portion. With this configuration, it is possible to
`
`selectively seed the celis only in the predetermined region of the permeable membrane
`
`by the 1 seal member and the 2 seal member.
`
`[0016]
`
`in this case, preferably, the other side discharge portion has a 1 other-side discharge
`
`port and a 1 other-side discharge port different from the 2 other-side discharge port, and
`
`a predetermined region includes a 1 region and a 1 region different from the 2 region,
`
`and the other side opening portion of the 2 seal memberis provided. A 1 side opening
`
`part exposing a 1 area of the permeabie membrane and a 2 side opening part exposing
`
`a 2 area of the permeable membraneare provided, and a cell includes a 1 cell anda 2
`
`cell, and a cell suspension introduced from the side introduction part is conducted to one
`
`side opening part of the 1 seal member. A 1 cell is seeded in a 4 region of the permeable
`
`membrane, and a liquid medium transmitted through the 1 region of the permeable
`
`membrane is conducted from the 1 other side opening portion to the 1 other side
`
`discharge port and is discharged from the 1 side discharge port. The 2 cell is seeded in
`
`the 2 region of the permeable membrane, and the liquid medium that has permeated
`
`through the 2 region of the permeable membrane is conducted from the 2 side opening
`
`to the 2 side outlet and discharged from the 2 side outlet. With this configuration, while
`
`flowing a cell suspension at a constant flow rate, cells can be filtered by beingfiltered
`
`

`

`through a predetermined region (1 region and 2 region) of the permeable membrane.
`
`That is, even when a plurality of cells (7 and 2 cells) are seeded in each of different
`
`regions, it is possible to reduce dead cells.
`
`f0017]
`
`in the cell culture apparatus having the 1 other-side discharge port and the 2 other-side
`
`discharge port, preferably, the other-side 7 flow passage member has a 1 other-side
`
`discharge port and a 2 other-side discharge port, and the other-side 2 flow passage
`
`member has a second other-side discharge port. A 1 side flow passage connecting the
`
`1 side opening part and the 1 side discharge port, and a 2 side passage connecting the
`
`2 side opening and the 2 side discharge port and separated from the 17 side flow passage
`
`are provided. With this configuration, since the 1 other-side flow channel and the 2 other-
`
`side flow channel are separated from each other, different media can be supplied to each
`
`of the flow channels. As a result, each of the 1 ceil and the 2 cell can be individually
`
`cultured by a medium suitable for each of the 1 ceil and the 2 cell.
`
`[001 8]
`
`A cell seeding method according to a 2 aspect of the present invention is a method of
`
`introducing a cell suspension from oneside introduction portion of a one side introduction
`
`portion and one side ejection portion which is arranged on one side of a permeable
`
`membrane which transmits a liquid medium without permeating a cell against a cell
`
`suspension in which cells are suspended in a liquid medium. A cell suspension
`
`introduced from one side introduction part via one side introduction part and one side
`
`flow passage which is arranged between the oneside introduction part and the one side
`
`discharge part and is communicated with one side introduction part and one side
`
`discharge part ;
`
`A step of passing through the permeable membrane, a step of seeding cells contained
`
`in the cell suspension circulated through the one side channel onto the permeable
`
`membrane, and transmitting the liquid medium contained in the cell suspension to the
`
`other side of the permeable membrane, and a step of circulating the liquid medium
`
`permeated through the permeable membrane through the other side channel arranged
`
`on the other side of the permeable membrane ; The method includes a step of
`
`discharging a liquid medium flowing through the other side flow path from the other side
`
`discharge section and the other-side side discharge section which are arranged on
`
`opposite sides of the other side flow path and which are arranged on the opposite side
`
`of the permeablefilm with respect to the other side flow path.
`
`[0019]
`
`

`

`in the cell seeding method according to the 2 aspect of the present invention, as
`
`described above, by introducing the cell suspension from the one side introduction
`
`portion and discharging the liquid medium from the other side discharge portion, the cell
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`suspension can be filtered by the permeation membrane while flowing the cell
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`suspension at a constant flow rate. As a result, unlike the case wherecells in a stationary
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`cell suspension are precipitated and seeded on a permeable membrane, it is possible to
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`suppress accumulation of cells in portions other than the permeable membrane. As a
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`result,
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`it is possible to reduce the dead cells due to the fact that the medium is not
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`supplied through the permeable membrane. Thus, it is possible to prevent the dead ceils
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`from adversely affecting the cells seeded on the permeable membrane, so that the ceils
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`can be appropriately cultured (analyzed).
`
`[Effect of the Invention]
`
`{0020}
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`According to the present invention, as described above,
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`it
`
`is possible to suppress
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`inappropriate cell culture (analysis) due to collapse of an environment simulating a living
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`body due to the fact that cells are not appropriately seeded on a transmission membrane.
`
`[Brief Description of the Drawings]
`
`[0021]
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`{[Fig. 1]FIlG. 7 is an exploded perspective view showing a configuration of a cell culture
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`apparatus (cell culture container) according to a first embodiment;.
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`[Fig. Z2]FIG. 1 is a plan view showing a configuration of a cell culture apparatus (cell
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`culture container) according to a first embodiment5.
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`[Fig. 3]FIG. 9 is a diagram for explaining a state in which cells are seeded on a permeable
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`membrane of a cell culture apparatus according to a 1 embodiment;.
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`{Fig. 4]FIG. 1 is a perspective view showing a configuration of a first seal memberof a
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`cell culture apparatus according to a 1 embodiment;.
`
`[Fig. 5]FIG. 1
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`is a diagram showing an overall configuration of a cell culture apparatus
`
`according to a first embodiment;.
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`[Fig. 6JFIG.
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`1
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`is a flow diagram illustrating a cell seeding method and a cell culture
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`method according to a first embodiment;.
`
`[Fig. 7]FIG. 2 is an exploded perspective view showing a configuration of a cell culture
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`apparatus (cell culture container) according to a first embodiment;.
`
`{[Fig. 8]FIG. 2 is a plan view showing a configuration of a cell culture apparatus (cell
`
`culture container) according to a first embodiment;.
`
`[Fig. 9]FIG. 9 is a diagram for explaining a state in which cells are seeded on a permeable
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`membrane of a cell culture apparatus according to a 2 embodiment;.
`
`

`

`[Fig. 10]FIG. 2 is a diagram showing an overall configuration of a cell culture apparatus
`
`according to a first embodiment;.
`
`{[Fig. 11]FIG. 1 is a schematic enlarged view of the vicinity of a permeable membrane
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`seeded with ceils of a cell culture apparatus according to a first embodiment;.
`
`[Mode for carrying out the invention]
`
`{0022}
`
`Hereinafter, the embodiment which materialized the present
`
`invention is described
`
`based on Drawings.
`
`{0023}
`
`{1 Embodiment]
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`Referring to FIGS. 1 to 6, a configuration of a cell culture apparatus 100 according to a
`1 embodiment will be described.
`
`[0024]
`
`The cell culture apparatus 100 is used for basic research in the field of drug discovery
`
`and biology, and is expected to be applied to thefield of regenerative medicine and the
`
`like. The cell culture apparatus 100 (see FIG. 11} is intended to reproduce an
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`environment in a living body by allowing a culture medium to flow through each of
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`channels (a channel 41 a and a channel 21 a described later) formed above and below
`
`a permeable membrane 1 described later. Specifically, in the 1 embodiment, cells 101
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`(in the 1 embodiment, intestina! epithelial cells} and goblet cells 102 are seeded on the
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`permeable membrane 1, and mucin layer 103 (see FIG. 11) is formed by the mucin
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`secreted by the goblet cells 102 (see FIG. 11). Thus, the cell culture apparatus 100 is
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`configured to be capable of reproducing an environment of an intestinal tract. In this case,
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`a channel 21 a, which will be described fater, reproduces an environmentin the intestinal
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`tract (in which intestinal bacteria or the like is present), and a channel 42 a, which will be
`
`described later, reproduces an environment outside the intestine (through which blood
`
`flows).
`
`{O025]
`
`(Configuration of Cell Culture Apparatus)
`
`Cell culture apparatus 100 includes cell culture vessel 100 A. The cell culture device 100
`
`(cell culture container 100 a) includes a permeable membrane 1. The permeable
`
`membrane 1
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`is configured to transmit the cell 101 through a liquid medium with respect
`
`to a cell suspension in which the cell 101 (see hatched portion in FIG. 3) is suspended
`
`in a liquid medium. Note that the permeable film 1 is, for example, a porousfilm.
`
`[0026]
`
`

`

`Further, the cell culture device 100 (cell culture container 100 a) includes a channel!
`
`member 2 disposed on the upper side (Z 4 direction side) of the permeable membrane
`
`7. The channel member2 includes a channel member 20 and a channel member 21
`
`disposed between the channel member 20 and the permeation membrane 1. Note that
`
`the channe! member 2 is an example of the "one side channe! member"in the claims.
`
`Further, each of the channel member 20 and the channel member 21 is an example of
`
`the "one side 1 channel member" and the “one side 2 channel member".
`
`[0027]
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`Each of the channel member 20 and the channe!] member21 is a plate-like member and
`
`has a square shape. The channel member 20 is made of glass, for example. The channel
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`member 21 is formed of, for example, silicone rubber. In addition, the length of each of
`
`the channel member 20, the channel member 21, the gasket 3, which will be described
`
`later, a later-described gasket 5, a channel member 40, which will be described later,
`
`and a chan