JP7107528B2 - cell culture vessel - Google Patents

Description

The present invention relates to a cell culture vessel for culturing cells therein and for measuring the electrical resistance of the cultured cells.

Techniques are known for measuring the electrical resistance of cells in order to investigate the properties of cultured cells and the state of culture. For example, in transepithelial electrical resistance (TEER) measurement, electrodes are placed on one side and the other side of a membrane for cell culture in a culture solution to measure the electrical resistance value. Measure the electrical resistance value of itself. A technique for measuring the electric resistance value of cells is described in Patent Document 1, for example.

On the other hand, so-called microfluidic devices are being developed. In the microfluidic device, by making the cell culture space a closed space except for fine channels, it is possible to perform cell culture while excluding external factors as much as possible. A technique for measuring the electric resistance value of cells in such a microfluidic device is described in Patent Document 2, for example.

WO2012/147463 Japanese Patent Publication No. 2017-513483

In the microfluidic device described in Patent Document 2, since the cell culture region and the electrodes are arranged at overlapping positions, it is difficult to observe the entire cultured cells. On the other hand, if the electrodes are arranged at positions distant from the cell culture region, the electrical resistance between the electrodes increases, and there is a risk that the accuracy of measuring the electrical resistance of the cells themselves will decrease.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a cell culture vessel that does not interfere with observation of cultured cells and that can accurately measure the electrical resistance of cells. .

In order to solve the above problems, the first invention of the present application is a cell culture vessel for culturing cells therein and for measuring the electrical resistance of the cultured cells, wherein the cell culture chamber is capable of storing a culture medium. and a cell culture membrane that divides the inside of the cell culture chamber into an upper space and a lower space between the bottom surface and the top surface of the cell culture chamber, and directly or indirectly between the outside and the cell culture chamber a first electrode chamber that directly communicates with the cell culture chamber above the cell culture membrane; and a second electrode chamber that directly communicates with the cell culture chamber below the cell culture membrane. a first working electrode and a first reference electrode arranged in the first electrode chamber; and a second working electrode and a second reference electrode arranged in the second electrode chamber; The body has a first member, a second member, a third member, a fourth member, a fifth member and a sixth member that overlap in the vertical direction, and the second member, the third member, the fourth member and the The fifth member has a through hole penetrating vertically, and the cell culture chamber includes the upper surface of the first member, the lower surface of the sixth member, the second member, the third member, and the fourth member. The first electrode chamber is formed by the upper surface of the fourth member, the lower surface of the sixth member, the inner surface of the fifth member, and the second electrode chamber. The electrode chamber is formed by the upper surface of the first member, the lower surface of the third member, and the inner surface of the second member, and at least part of each of the first electrode chamber and the second electrode chamber is The cell culture membrane does not vertically overlap the cell culture region, which is the portion inside the cell culture chamber.

The second invention of the present application is the cell culture vessel of the first invention, wherein the minimum cross-sectional area of the space between the first working electrode and the cell culture region and the space between the second working electrode and the cell culture region is larger than the maximum channel area of the channel.

A third invention of the present application is the cell culture vessel according to the first invention or the second invention, wherein the housing constituting the cell culture chamber is transparent at a position vertically overlapping at least a part of the cell culture area. It has a portion made of material.

A fourth invention of the present application is the cell culture vessel according to any one of the first to third inventions, wherein the surface area of the first working electrode in the first electrode chamber is equal to that of the first reference electrode. The surface area of the second working electrode within the second electrode chamber is greater than the surface area of the second working electrode within the second electrode chamber than the surface area of the second reference electrode within the second electrode chamber.

A fifth invention of the present application is the cell culture vessel according to any one of the first invention to the fourth invention, wherein the first electrode chamber is positioned in one direction perpendicular to the vertical direction with respect to the cell culture chamber. and the second electrode chamber is arranged on the other side in the first direction with respect to the cell culture chamber.
A sixth invention of the present application is the cell culture vessel according to the fifth invention, wherein the first working electrode, the cell culture region and the second working electrode are arranged on a straight line, and the first working electrode and the The housing is not arranged on a straight line connecting the second working electrode.
A seventh invention of the present application is the cell culture vessel according to the sixth invention, wherein the first reference electrode, the cell culture region, and the second reference electrode are arranged on a straight line, and the first reference electrode and the The housing is not arranged on a straight line connecting the second reference electrode.

According to the first to seventh inventions of the present application, it is possible to prevent the electrode from overlapping the cell culture region and interfering with observation. In addition, it is possible to suppress a decrease in the measurement accuracy of the electrical resistance of cells due to an increase in the electrical resistance between the electrodes.

In particular, according to the second invention of the present application, the influence of the external environment in the cell culture chamber can be reduced by reducing the channel area of each channel. In addition, it is easy to control the inflow and outflow of the culture solution and reagents into the cell culture chamber. On the other hand, by increasing the minimum cross-sectional area of the space between each electrode and the cell culture area, the electrical resistance of the culture solution between each electrode and the cell culture area is reduced. This makes it possible to measure the electrical resistance of cells with higher accuracy.

In particular, according to the third invention of the present application, it is easy to observe the state of cells cultured in the cell culture area.

In particular, according to the fifth invention of the present application, the electrical resistance of the culture solution between the electrodes becomes smaller. Thereby, the electrical resistance of cells can be measured with higher accuracy.
In particular, according to the sixth invention of the present application, the electrical resistance of the culture solution between the first working electrode and the second working electrode becomes smaller. Thereby, the electrical resistance of cells can be measured with higher accuracy.
In particular, according to the seventh invention of the present application, the electric resistance of the culture solution between the first reference electrode and the second reference electrode is further reduced. Thereby, the electrical resistance of cells can be measured with higher accuracy.

1 is a vertical cross-sectional view of a cell culture vessel according to a first embodiment; FIG. 1 is a plan view of a cell culture vessel according to a first embodiment; FIG. 1 is an exploded perspective view of a cell culture vessel according to a first embodiment; FIG. 4 is a schematic diagram showing electrical connections of the measurement unit according to the first embodiment; FIG. FIG. 4 is a vertical cross-sectional view of a cell culture vessel according to a second embodiment; FIG. 10 is a plan view of the cell culture vessel according to the second embodiment. FIG. 4 is an exploded perspective view of a cell culture vessel according to a second embodiment; FIG. 10 is a layout diagram of a cell culture vessel according to a modified example as viewed from the top. FIG. 11 is a top view layout diagram of a cell culture vessel according to another modified example.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, the direction parallel to the bottom surface of the cell culture vessel is called "horizontal direction", and the direction orthogonal to the horizontal direction is called "vertical direction". However, when using the cell culture vessel, the bottom surface of the cell culture vessel does not have to be horizontal.

<1. First Embodiment>
<1-1. Configuration of Cell Culture Vessel>
FIG. 1 is a longitudinal sectional view of a cell culture vessel 1 according to a first embodiment of the invention. FIG. 2 is a layout diagram showing the layout of each part in the top view of the cell culture vessel 1. As shown in FIG. FIG. 3 is an exploded perspective view of the cell culture vessel 1. FIG.

The cell culture vessel 1 is a vessel for culturing cells therein and for measuring the electrical resistance of the cultured cells. This cell culture vessel 1 is a so-called microfluidic device in which the internal space is a closed space except for fine flow paths 24 , 25 , 26 and 27 .

As shown in FIGS. 1 and 2, the cell culture vessel 1 has a cell culture chamber 21, a first electrode chamber 22, a second electrode chamber 23, a first upper channel 24, a second It has an upper channel 25 , a first lower channel 26 and a second lower channel 27 . That is, the main body part 10 is a housing that constitutes the cell culture chamber 21 . The cell culture vessel 1 also has a first working electrode 31 , a first reference electrode 32 , a second working electrode 41 and a second reference electrode 42 .

As shown in FIGS. 1 and 3, the main body 10 of the cell culture vessel 1 includes a first member 11, a second member 12, a third member 13, a fourth member 14, and a first member 11, a second member 12, a third member 13, a fourth member 14, and a third member 11, which are vertically stacked. It is composed of the 5th member 15 , the 6th member 16 and the cell culture membrane 20 . The body portion 10 includes a first member 11, a second member 12, a third member 13, a cell culture membrane 20, a fourth member 14, a fifth member 15, and a sixth member 16 in order from the bottom to the top. It is composed by overlapping.

The cell culture chamber 21 is arranged in the center of the cell culture vessel 1 . In this embodiment, the cell culture chamber 21 is formed by the upper surface of the first member 1, the lower surface of the sixth member 16, and the inner surfaces of the second to fifth members 12-15. A culture solution can be stored in the cell culture chamber 21 . The cell culture membrane 20 is vertically spaced from each of the bottom surface and top surface of the cell culture chamber 21 . Thereby, the cell culture membrane 20 partitions the inside of the cell culture chamber 21 into an upper space and a lower space between the top surface and the bottom surface of the cell culture chamber 21 .

The cell culture membrane 20 is a membrane having cell adhesiveness. For the cell culture membrane 20 of this embodiment, for example, a thin film called a membrane having a large number of fine through holes is used. Materials for the membrane include, for example, PC (polycarbonate), PTFE (polytetrafluoroethylene), PET (polyethylene terephthalate), and the like. The surface of the membrane may be coated with collagen or the like to enhance cell adhesion. A portion of the cell culture membrane 20 exposed inside the cell culture chamber 21 serves as a cell culture region 200 . In the present embodiment, portions of the cell culture membrane 20 other than the cell culture region 200 are covered with the third member 13 and the fourth member 14 and are not exposed. Instead of the cell culture membrane 20, a plate-like member having cell adhesiveness and provided with a large number of fine through holes may be used.

The first electrode chamber 22 directly communicates with the cell culture chamber 21 above the cell culture membrane 20 . The first electrode chamber 22 is arranged on one side in the first direction with respect to the cell culture chamber 21 . In this embodiment, the upper surface of the fourth member 14 , the lower surface of the sixth member 16 and the inner surface of the fifth member 15 form the first electrode chamber 22 .

The second electrode chamber 23 directly communicates with the cell culture chamber 21 below the cell culture membrane 20 . The second electrode chamber 23 is arranged on the other side in the first direction with respect to the cell culture chamber 21 . In this embodiment, the upper surface of the first member 11, the lower surface of the third member 13, and the inner surface of the second member 12 form the second electrode chamber 23. As shown in FIG. Moreover, the first electrode chamber 22 and the second electrode chamber 23 do not overlap the cell culture region 200 in the vertical direction.

The “first direction” is a direction parallel to the bottom surface of the cell culture vessel 1 and perpendicular to the vertical direction. 1 and 2, the first direction is the horizontal direction of the paper, the one side of the first direction is the right side of the paper, and the other side of the first direction is the left side of the paper. Moreover, the “second direction” is a direction parallel to the bottom surface of the cell culture vessel 1 and orthogonal to the first direction. In FIG. 2, the second direction is the vertical direction of the paper, the one side of the second direction is the upper side of the paper, and the other side of the second direction is the lower side of the paper.

The first upper channel 24 and the second upper channel 25 are channels that connect the outside of the cell culture container 1 and the cell culture chamber 21 above the cell culture membrane 20 . One end of the first upper channel 24 communicates directly with the cell culture chamber 21 , and the other end opens to the end surface of the cell culture vessel 1 on one side in the second direction. One end of the second upper channel 25 communicates directly with the cell culture chamber 21 , and the other end opens to the end surface of the cell culture vessel 1 on the other side in the second direction. In this embodiment, the upper surface of the fourth member 14 , the lower surface of the sixth member 16 , and the inner surface of the fifth member 15 form a first upper flow path 24 and a second upper flow path 25 .

The first lower channel 26 and the second lower channel 27 are channels that communicate the outside of the cell culture vessel 1 and the cell culture chamber 21 below the cell culture membrane 20 . One end of the first lower channel 26 communicates directly with the cell culture chamber 21 , and the other end opens to the end surface of the cell culture vessel 1 on one side in the second direction. One end of the second lower channel 27 communicates directly with the cell culture chamber 21 , and the other end opens to the end surface of the cell culture vessel 1 on the other side in the second direction. In this embodiment, the upper surface of the first member 11, the lower surface of the third member 13, and the inner surface of the second member form a first lower flow path 26 and a second lower flow path 27. As shown in FIG. In this embodiment, the first upper flow path 24 and the first lower flow path 26 overlap in the vertical direction, and the second upper flow path 25 and the second lower flow path 27 overlap in the vertical direction. placed.

A first working electrode 31 and a first reference electrode 32 are arranged in the first electrode chamber 22 . Specifically, the first working electrode 31 and the first reference electrode 32 are fixed to the top surface of the first electrode chamber 22 . The first working electrode 31 and the first reference electrode 32 are formed, for example, by applying an electrode paste, which is the material of the electrodes, to the lower surface of the sixth member 16 that constitutes the top surface of the first electrode chamber 22 and drying it. be. The first working electrode 31 and the first reference electrode 32 may be formed by evaporating metal on the top surface of the first electrode chamber 22, or plate-like electrodes may be formed on the top surface of the first electrode chamber 22. may be adhered to.

A second working electrode 41 and a second reference electrode 42 are arranged in the second electrode chamber 23 . Specifically, the second working electrode 41 and the second reference electrode 42 are fixed to the bottom surface of the second electrode chamber 23 . The second working electrode 41 and the second reference electrode 42 are formed, for example, by applying an electrode paste, which is the material of the electrodes, to the upper surface of the first member 11 that forms the bottom surface of the second electrode chamber 23 and drying it. . The second working electrode 41 and the second reference electrode 42 may be formed by vapor-depositing metal on the bottom surface of the second electrode chamber 23, or a plate-like electrode may be adhered to the bottom surface of the second electrode chamber 23. It may be

In this way, by arranging at least a part of the first electrode chamber 22 and the second electrode chamber 23 for arranging the electrodes at a position not vertically overlapping the cell culture region 200, the electrodes 31, 32, 41 , 42 overlap with the cell culture region 200 and interfere with observation.

In addition, by providing the first electrode chamber 22 and the second electrode chamber 23 at positions adjacent to the cell culture chamber 21, it is possible to prevent the distance between the cell culture region 200 and the electrodes 31, 32, 41, 42 from increasing. can. Therefore, it is possible to prevent the electrical resistance between the working electrodes 31 and 41 and between the reference electrodes 32 and 42 from increasing and lowering the measurement accuracy of the cell electrical resistance.

In this embodiment, the minimum cross-sectional area of the space between the first working electrode 31 and the cell culture region 200 is larger than the maximum flow area of each of the channels 24-27. Also, the minimum cross-sectional area between the second working electrode 41 and the cell culture region 200 is larger than the maximum flow area of each of the flow paths 24-27. In this way, by reducing the channel area of each of the channels 24 to 27, the influence of the external environment inside the cell culture chamber 21 can be reduced. In addition, it is easy to control the inflow and outflow of the culture solution and reagents to the cell culture chamber 21 . On the other hand, when the minimum cross-sectional area of the space between each electrode 31, 32, 41, 42 and the cell culture region 200 is larger, the culture solution between each electrode 31, 32, 41, 42 and the cell culture region 200 electrical resistance becomes smaller. Thereby, the electric resistance of the cells cultured on the cell culture area 200 can be measured with higher accuracy.

Further, in the present embodiment, the first electrode chamber 22 is arranged on one side in the first direction with respect to the cell culture chamber 21, and the second electrode chamber 23 is arranged on the other side in the first direction with respect to the cell culture chamber 21. be. That is, the first electrode chamber 22 and the second electrode chamber 23 are arranged at positions facing each other with the cell culture chamber 21 interposed therebetween. Thereby, the first working electrode 31, the cell culture region 200, and the second working electrode 41 are arranged substantially on a straight line. Also, the first reference electrode 32, the cell culture region 200, and the second reference electrode 42 are arranged substantially on a straight line. Therefore, the electrical resistance of the culture solution between the first working electrode 31 and the second working electrode 41 and the electrical resistance of the culture solution between the first reference electrode 32 and the second reference electrode 42 become smaller. Thereby, the electric resistance of the cells cultured on the cell culture area 200 can be measured with higher accuracy.

As shown in FIG. 2 , in this embodiment, the surface area of the first working electrode 31 within the first electrode chamber 22 is greater than the surface area of the first reference electrode 32 within the first electrode chamber 22 . Also, the surface area of the second working electrode 41 in the second electrode chamber 23 is larger than the surface area of the second reference electrode 42 in the second electrode chamber 23 .

In order to increase the electric capacitance on the surfaces of the working electrodes 31, 41, it is preferable to design the surface areas of the working electrodes 31, 41 as large as possible. Arranging the electrodes 31 , 32 , 41 , 42 in the electrode chambers 22 , 23 increases the degree of freedom in designing the electrodes 31 , 32 , 41 , 42 . Therefore, as in the present embodiment, the working electrodes 31 and 41 can be designed to have relatively large surface areas. Further, by arranging the electrodes 31, 32, 41, 42 in the electrode chambers 22, 23, even when the surface areas of the working electrodes 31, 41 are increased, the electrodes 31, 32, 41, 42 are It is difficult to overlap with the cell culture area 200 in the vertical direction.

In this embodiment, the members 11 to 16 forming the main body 10 are made of PDMS (polydimethylsiloxane). PDMS has high transparency and low autofluorescence. Therefore, it is easy to observe cells on the cell culture area 200 from the outside of the main body 10 . In the present embodiment, all portions of the main body 10 that overlap the cell culture region 200 in the vertical direction are made of PDMS, which is a transparent material. However, it is sufficient that the main body 10 has a portion formed of a transparent material at a position vertically overlapping at least a portion of the cell culture region 200 . For example, the cell culture area 200 can be observed if a part of the upper side of the cell culture area 200 is made of a transparent material.

<1-2. Electrical resistance measurement of cultured cells>
Next, a method for measuring the electrical resistance of cultured cells using the cell culture vessel 1 will be described with reference to FIG. FIG. 4 is a schematic diagram showing electrical connections of the measurement unit 9. As shown in FIG. Note that FIG. 4 shows the electrical connection of the measurement unit 9 when electrical resistance is measured for one cell culture vessel 1 . In addition, in the actual electrical resistance measurement, there are cases where the measurement is performed for a plurality of cell culture vessels 1 at the same time. In that case, for example, the connection destination of the power supply device 91 and the voltmeter 92 of the measuring device 90 to be described later can be switched for each cell culture vessel 1 .

As shown in FIG. 4 , the measurement unit 9 is composed of the cell culture vessel 1 and the culture solution in the cell culture vessel 1 and a measurement device 90 connected to the cell culture vessel 1 .

The measuring device 90 has a power supply device 91 and a voltmeter 92 . Two output terminals of the power supply device 91 are connected to the first working electrode 31 and the second working electrode 41 . Also, two input terminals of the voltmeter 92 are connected to the first reference electrode 32 and the second reference electrode 42 . When measuring the electrical resistance of cultured cells, the cell culture container 1 is filled with a culture solution.

In FIG. 4, the resistance Rm is the electrical resistance between the cell culture region 200 and cells cultured on the cell culture region 200 (hereinafter referred to as “cell portion”). A resistance Rw1 is the electrical resistance of the culture solution between the first working electrode 31 and the cell portion. A resistance Rw2 is the electrical resistance of the culture solution between the second working electrode 41 and the cell portion. A resistance Rr1 is the electrical resistance of the culture solution between the first reference electrode 32 and the cell portion. A resistance Rr2 is the electrical resistance of the culture solution between the second reference electrode 42 and the cell portion.

The resistance values of the resistances Rw1, Rr1, Rw2, and Rr2 between the electrodes 31, 32, 41, and 42 and the cell portion and the resistance Rm of the cell culture region 200 in which no cells are cultured are set in advance. measured as a control.

Then, the power supply device 91 is driven to apply a constant current between the first working electrode 31 and the second working electrode 41 . At the same time, the voltmeter 92 measures the voltage value between the first reference electrode 32 and the second reference electrode 42 . Since the working electrodes 31 and 41 and the reference electrodes 32 and 42 are arranged sufficiently close to each other, the current flowing between the reference electrodes 32 and 42 and the current flowing between the working electrodes 31 and 41 have the same current value. can be approximated as Therefore, the electrical resistance value between the reference electrodes 32 and 42 is calculated from the voltage value between the reference electrodes 32 and 42 . Furthermore, the resistance Rm of the cell part is calculated based on each value measured in advance. Thereby, the electrical characteristics of the cells cultured on the cell culture region 200 can be obtained.

When a potential is applied between the first working electrode 31 and the second working electrode 41 using the power supply device 91, an electric double layer is formed on the electrode surfaces of the first working electrode 31 and the second working electrode 41. be. At this time, the output potential from the power supply device 91 is the potential applied to the entire circuit. Therefore, the output potential from the power supply device 91 does not have an accurate voltage value between the working electrodes 31 and 41 . Therefore, a first reference electrode 32 and a second reference electrode 42 are placed in the vicinity of the first working electrode 31 and the second working electrode 41, respectively, and the potential between the reference electrodes 32 and 42 is measured. By calculating using the measured potential, the resistance Rm of the cell part can be measured more accurately.

<2. Second Embodiment>
Next, a cell culture vessel 1A according to a second embodiment of the present invention will be described with reference to FIGS. 5 and 6. FIG. FIG. 5 is a longitudinal sectional view of the cell culture vessel 1A. FIG. 6 is a layout diagram showing the layout of each part in the top view of the cell culture vessel 1A. FIG. 7 is an exploded perspective view of the main body 10A of the cell culture vessel 1A.

As shown in FIGS. 5 and 6, the cell culture vessel 1A, like the cell culture vessel 1 according to the first embodiment, has a cell culture chamber 21A, a first electrode chamber 22A, a second It has two electrode chambers 23A, a first upper channel 24A, a second upper channel 25A, a first lower channel 26A, and a second lower channel 27A. The cell culture vessel 1A also has a first working electrode 31A, a first reference electrode 32A, a second working electrode 41A, and a second reference electrode 42A.

As shown in FIGS. 5 and 7, the main body 10A of the cell culture vessel 1A includes a first resin member 11A, a second member 12A, a third member 13A, a fourth member 14A, and a third member 11A, which are vertically stacked. It is composed of the fifth member 15A, the sixth member 16A, and the cell culture membrane 20A. The body portion 10A includes, from the bottom to the top, a first member 11A, a second member 12A, a third member 13A, a cell culture membrane 20A, a fourth member 14A, a fifth member 15A, and a sixth member 16A in this order. It is composed by overlapping.

The arrangement of the cell culture chamber 21A, the first electrode chamber 22A, the second electrode chamber 23A, the channels 24A, 25A, 26A, 27A, and the cell culture area 200A is similar to that of the cell culture chamber 1 of the cell culture vessel 1 according to the first embodiment. 21 , the first electrode chamber 22 , the second electrode chamber 23 , the channels 24 , 25 , 26 , 27 and the cell culture area 200 .

The first working electrode 31A, the first reference electrode 32A, the second working electrode 41A and the second reference electrode 42A are cylindrical electrodes extending vertically. The body portion 10A has two first through holes 51A extending from the top surface of the body portion 10A to the top surface of the first electrode chamber 22A, and two second through holes extending from the top surface of the body portion 10A to the top surface of the second electrode chamber 23A. and a through hole 52A.

The first working electrode 31A and the first reference electrode 32A are each inserted into the first through hole 51A. The upper end portions of the first working electrode 31A and the first reference electrode 32A are respectively arranged above the upper surface of the main body portion 10A. Also, the lower ends of the first working electrode 31A and the first reference electrode 32A are arranged in the first electrode chamber 22A.

The second working electrode 41A and the second reference electrode 42A are each inserted into the second through hole 52A. The upper end portions of the second working electrode 41A and the second reference electrode 42A are respectively arranged above the upper surface of the main body portion 10A. Also, the lower ends of the second working electrode 41A and the second reference electrode 42A are respectively arranged in the second electrode chamber 23A.

In this way, each of the electrodes 31A, 32A, 41A, 42A may be arranged so as to pass through the body portion 10A.

<3. Variation>
Although the main embodiments of the present invention have been described above, the present invention is not limited to the above embodiments.

FIG. 8 is a layout diagram showing the layout of each part in a top view of a cell culture vessel 1B according to one modification. As shown in FIG. 8, the cell culture vessel 1B, like the cell culture vessel 1 according to the first embodiment, has a cell culture chamber 21B, a first electrode chamber 22B, and a second electrode chamber as internal spaces of the main body 10B. 23B, a first upper channel 24B, a second upper channel 25B, a first lower channel 26B, and a second lower channel 27B. Moreover, the cell culture vessel 1B has a first working electrode 31B, a first reference electrode 32B, a second working electrode 41B, and a second reference electrode 42B.

The arrangement of the cell culture chamber 21B, the first electrode chamber 22B, the second electrode chamber 23B, the cell culture area 200B, and the electrodes 31B, 32B, 41B, and 42B is similar to that of the cell culture chamber 21 of the cell culture vessel 1 according to the first embodiment. , the first electrode chamber 22 , the second electrode chamber 23 , the cell culture area 200 , and the electrodes 31 , 32 , 41 , 42 .

In the first embodiment and the second embodiment described above, each channel is arranged substantially in the center of the cell culture chamber in the first direction. That is, each channel directly communicated with substantially the center of the cell culture chamber in the first direction. However, the present invention is not limited to this.

In the example of FIG. 8, the first upper flow path 24B is communicated with the cell culture chamber 21B on one side of the center in the first direction. The second upper flow path 25B communicates with the cell culture chamber 21B on the other side of the center in the first direction. The first lower channel 26B is connected to the cell culture chamber 21B on the other side of the center in the first direction. Also, the second lower channel 27B is connected to the cell culture chamber 21B on one side of the center in the first direction.

As a result, the inflow position and the outflow position of the culture solution are different between the upper side and the lower side of the cell culture region 200B when viewed from the top. As in the example of FIG. 8, the positions of the channels 24B, 25B, 26B, and 27B can be changed as appropriate.

FIG. 9 is a layout diagram showing the layout of each part in a top view of a cell culture vessel 1C according to one modification. As shown in FIG. 9, the cell culture vessel 1C, like the cell culture vessel 1 according to the first embodiment, has a cell culture chamber 21C, a first electrode chamber 22C, and a second electrode chamber as internal spaces of the main body 10C. 23C, a first upper channel 24C, a second upper channel 25C, a first lower channel 26C, and a second lower channel 27C. The cell culture vessel 1C also has a first working electrode 31C, a first reference electrode 32C, a second working electrode 41C, and a second reference electrode 42C.

The cell culture chamber 21C, the first electrode chamber 22C, the second electrode chamber 23C, the cell culture area 200C, and the electrodes 31C, 32C, 41C, and 42C are arranged in the cell culture chamber 21 of the cell culture vessel 1 according to the first embodiment. , the first electrode chamber 22 , the second electrode chamber 23 , the cell culture area 200 , and the electrodes 31 , 32 , 41 , 42 .

In the above embodiments and variations, each flow channel communicated directly with the cell culture chamber. However, the present invention is not limited to this.

In the example of FIG. 9, the first upper flow path 24C is connected to the first electrode chamber 22C on one side in the first direction of the cell culture chamber 21C. Thereby, the first upper flow path 24C indirectly communicates with the cell culture chamber 21C via the first electrode chamber 22C. The second upper flow path 25C directly communicates with the cell culture chamber 21C on the other side of the center in the first direction. The first lower channel 26C is connected to the second electrode chamber 23C on the other side in the first direction of the cell culture chamber 21C. Thereby, the first lower channel 26C indirectly communicates with the cell culture chamber 21C via the second electrode chamber 23C. Also, the second lower flow path 27C directly communicates with the cell culture chamber 21C on one side of the center in the first direction.

As a result, the inflow position and outflow position of the culture solution are different between the upper side and the lower side of the cell culture area 200C in top view. As in the example of FIG. 9, the channels 24C, 25C, 26C, and 27C do not necessarily communicate directly with the cell culture chamber, and may communicate indirectly.

Moreover, in the above embodiment, the shape of the cell culture vessel is a rectangular parallelepiped, but the present invention is not limited to this. The shape of the cell culture vessel can be appropriately designed into a columnar shape, a polygonal prismatic shape, a hemispherical shape, and other shapes.

Moreover, in the above-described embodiments, the shape of the cell culture region is substantially rectangular when viewed from above, but the present invention is not limited to this. The shape of the cell culture region may be circular, elliptical, polygonal, or any other complicated shape imitating a biological structure when viewed from above. In addition, although the cell culture areas are arranged horizontally in the above embodiments, the present invention is not limited to this. The cell culture area may have a slope as a whole in the horizontal direction, or may have a complicated shape in which the slope changes partially.

Further, the detailed configuration of the cell culture vessel may be different from each drawing of the present application. Also, the elements appearing in the above embodiments and modifications may be appropriately combined as long as there is no contradiction.

1, 1A, 1B, 1C cell culture vessel 10, 10A, 10B, 10C body 20, 20A cell culture membrane 21, 21A, 21B, 21C cell culture chamber 22, 22A, 22B, 22C first electrode chamber 23, 23A, 23B, 23C second electrode chamber 24, 24A, 24B, 24C first upper channel 25, 25A, 25B, 25C second upper channel 26, 26A, 26B, 26C first lower channel 27, 27A, 27B, 27C Second lower channel 31, 31A, 31B, 31C First working electrode 32, 32A, 32B, 32C First reference electrode 41, 41A, 41B, 41C Second working electrode 42, 42A, 42B, 42C Second reference Electrodes 200, 200A, 200B, 200C Cell culture area

Claims (7)

A cell culture vessel for culturing cells inside and measuring the electrical resistance of the cultured cells,
a cell culture chamber capable of storing a culture solution;
a cell culture membrane that divides the interior of the cell culture chamber into an upper space and a lower space between the bottom surface and the top surface of the cell culture chamber;
a channel that directly or indirectly communicates the outside with the cell culture chamber;
a first electrode chamber directly communicating with the cell culture chamber above the cell culture membrane;
a second electrode chamber directly communicating with the cell culture chamber below the cell culture membrane;
a first working electrode and a first reference electrode disposed within the first electrode chamber;
a second working electrode and a second reference electrode disposed within the second electrode chamber;
has
The housing has a first member, a second member, a third member, a fourth member, a fifth member and a sixth member that overlap in the vertical direction,
The second member, the third member, the fourth member and the fifth member have through holes that penetrate vertically,
The cell culture chamber is formed by the upper surface of the first member, the lower surface of the sixth member, and the inner surfaces of the second member, the third member, the fourth member and the fifth member,
the first electrode chamber is formed by the upper surface of the fourth member, the lower surface of the sixth member, and the inner surface of the fifth member;
the second electrode chamber is formed by the upper surface of the first member, the lower surface of the third member, and the inner surface of the second member;
The cell culture vessel, wherein at least a portion of each of the first electrode chamber and the second electrode chamber does not vertically overlap a cell culture region, which is a portion of the cell culture membrane inside the cell culture chamber. The cell culture vessel according to claim 1,
The minimum cross-sectional area of the space between the first working electrode and the cell culture region and the minimum cross-sectional area of the space between the second working electrode and the cell culture region are larger than the maximum flow channel area of the channel. , cell culture vessel. The cell culture vessel according to claim 1 or claim 2,
A cell culture container, wherein a housing constituting the cell culture chamber has a portion formed of a transparent material at a position vertically overlapping at least a part of the cell culture area. The cell culture vessel according to any one of claims 1 to 3,
the surface area of the first working electrode within the first electrode chamber is greater than the surface area of the first reference electrode within the first electrode chamber;
The cell culture vessel, wherein the surface area of the second working electrode in the second electrode chamber is larger than the surface area of the second reference electrode in the second electrode chamber. The cell culture vessel according to any one of claims 1 to 4,
The first electrode chamber is arranged on one side of the cell culture chamber in a first direction perpendicular to the vertical direction,
The cell culture container, wherein the second electrode chamber is arranged on the other side in the first direction with respect to the cell culture chamber. The cell culture vessel according to claim 5,
The first working electrode, the cell culture region and the second working electrode are arranged on a straight line,
The cell culture vessel, wherein the housing is not arranged on a straight line connecting the first working electrode and the second working electrode. The cell culture vessel according to claim 6,
The first reference electrode, the cell culture region and the second reference electrode are arranged on a straight line,
A cell culture vessel, wherein the housing is not arranged on a straight line connecting the first reference electrode and the second reference electrode.

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