Culture Device

Abstract

To provide a technique of effectively applying an electric current to a wide area of a measurement chamber while preventing electrodes from becoming an obstacle in observing a cultured product.SOLUTION: A culture device 1 is applicable for measurement of electric resistance of a cell 9. The culture device 1 has a measurement chamber 100, a permeation layer 30, an upper working electrode 40, and a lower working electrode 50. In the measurement chamber 100, the length in a first direction is longer than the length in a second direction crossing the first direction. The permeation layer 30 partitions the measurement chamber 100 into an upper first chamber 110 and a lower second chamber 120. The permeation layer 30 is capable of allowing liquid to pass. The upper working electrode 40 is positioned above the measurement chamber 100, and has working electrode parts 41, 42 whose length in the first direction is longer than the length in a second direction. The lower working electrode 50 is positioned above the measurement chamber 100, and has working electrode parts 51, 52 whose length in the first direction is longer than the length in the second direction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The subject matter disclosed herein relates to a culture device. [Background technology]

[0002] Techniques for measuring the electrical resistance of cultured materials, such as cultured cells, are known to examine their properties and culture conditions. For example, in transepithelial electrical resistance (TEER) measurements, electrodes are placed on one side and the other side of a cell culture membrane in a culture medium, and the electrical resistance between the electrodes is measured, thereby measuring the electrical resistance of cells cultured on the membrane. Such a technique for measuring the electrical resistance of cells is described, for example, in Patent Document 1.

[0003] Furthermore, Patent Document 2 discloses a microfluidic device in which a permeable membrane for culturing cells is disposed in a flow channel and multiple pairs of electrodes are disposed above and below the permeable membrane. In Patent Document 2, the upper and lower working electrodes extend in the short direction of the flow channel and are disposed so as to cross the flow channel in the short direction. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-137307 [Patent Document 2] Japanese Patent Publication No. 2020-146015 Summary of the Invention [Problem to be solved by the invention]

[0005] In TEER measurement, it is desirable to apply a uniform current to the entire culture in the measurement chamber. When electrodes are arranged in the short direction of the flow channel, as in Patent Document 2, it is possible to increase the width of the electrodes in the longitudinal direction of the flow channel in order to apply a current to a wide area within the flow channel. However, in this case, the increased width of the electrodes may interfere with observation of the culture. It is also possible to arrange multiple electrodes in the longitudinal direction of the flow channel in order to apply a current to a wide area within the flow channel. However, even in this case, the increased number of electrodes may interfere with observation of the culture.

[0006] An object of the present invention is to provide a technique for effectively applying a current over a wide area within a measurement chamber while preventing the electrodes from interfering with the observation of a culture. [Means for solving the problem]

[0007] In order to achieve the above object, a first aspect of the present invention is a culture device applicable to measuring the electrical resistance of a culture, the culture device including: a measurement chamber whose length in a first direction is longer than its length in a second direction intersecting the first direction; a permeable layer that separates the measurement chamber into a first chamber on one side and a second chamber on the other side in a third direction intersecting the first and second directions, the permeable layer allowing a liquid to pass through; a first working electrode that is located on one side of the measurement chamber in the third direction and has an electrode portion whose length in the first direction is longer than its length in the second direction; and a second working electrode that is located on the other side of the measurement chamber in the third direction and has an electrode portion whose length in the first direction is longer than its length in the second direction. a one-side reference electrode located on one side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction; Equipped with the one-side working electrode has a one-side first working electrode portion extending in the first direction, a one-side second working electrode portion extending in the first direction and positioned apart from the one-side first working electrode portion in the second direction, and a working bus portion to which one ends of the one-side first working electrode portion and the one-side second working electrode portion are connected; the one-side reference electrode has a one-side reference electrode portion positioned between the one-side first working electrode portion and the one-side second working electrode portion, the width of the one-side reference electrode portion being smaller than the widths of the one-side first working electrode portion and the one-side second working electrode portion, and the one-side first working electrode portion and the one-side second working electrode portion extending to the outside of the measurement chamber in the second direction. .

[0008] A second aspect is the culture device of the first aspect, wherein the other-side working electrode extends in the first direction and has a second-side first working electrode portion facing the one-side working electrode in the measurement chamber.

[0009] A third aspect is a culture device according to the first or second aspect, wherein the other-side working electrode extends in the first direction and has an other-side second working electrode portion located apart from the one-side working electrode in the second direction within the measurement chamber.

[0011] No. 4 The aspect is 3 In a culture device according to an embodiment, the other-side working electrode has an other-side first working electrode portion extending in the first direction and an other-side second working electrode portion extending in the first direction and positioned away from the other-side first working electrode portion in the second direction.

[0012] No. 5 The aspect is 4 In the culture device of the embodiment, the one-side first working electrode section faces the other-side first working electrode section in the measurement chamber.

[0013] No. 6 The aspect is 5 In the culture device of the embodiment, the one-side second working electrode section faces the other-side second working electrode section in the measurement chamber.

[0014] No. 7 The aspect is 4 From the aspect 6 In any one of the culture devices of the aspects, when the distance between the first working electrode portion on one side and the first working electrode portion on the other side is defined as a first distance, and the distance between the first working electrode portion on one side and the second working electrode portion on the other side is defined as a second distance, the second distance is 1.5 times or less of the first distance.

[0015] No. 8 The embodiments are the first to second embodiments. 7 The culture device of any one of the aspects further comprises a one-side reference electrode located on one side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction, and a other-side reference electrode located on the other side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction.

[0016] No. 9 The embodiments are the first to second embodiments. 8 The culture device of any one of the aspects further comprises a first member having an inner surface that forms the first chamber, a second member located on the other side of the first member in the third direction and having an inner surface that forms the second chamber, a one-side electrode substrate located on one side of the first member in the third direction and having a surface on which the one-side working electrode is arranged, and a other-side electrode substrate located on the other side of the second member in the third direction and having a surface on which the other-side working electrode is arranged, and the permeation layer is arranged between the first member and the second member.

[0017] No. 10 The embodiments are the first to second embodiments. 9 In the culture device of any one of the aspects, the one-side working electrode has an electrode portion extending from one end of the measurement chamber to the other end in the first direction, and the other-side working electrode has 1st direction The measuring chamber has an electrode portion extending from one end to the other end. [Effects of the Invention]

[0018] First to second aspects 10 In the culture device of this embodiment, the one-side working electrode is disposed relative to the measurement chamber so as to extend in the longitudinal direction of the measurement chamber, thereby preventing the one-side working electrode from interfering with observation of the culture, and enabling current to be effectively applied to a wide area within the measurement chamber. Furthermore, the range in which the current is applied can be expanded in the second direction.

[0019] According to the culture device of the second aspect, the other-side second working electrode section faces the one-side working electrode section, so that a current can be effectively applied to the culture between the one-side working electrode section and the other-side working electrode section.

[0020] According to the culture device of the third aspect, the other-side second working electrode section is disposed apart from the one-side working electrode in the second direction, so that the range in which the current flows can be widened in the second direction.

[0022] No. 4 According to the culture device of the embodiment, the range in which the current flows can be expanded in the second direction.

[0023] No. 5 According to the culture device of the embodiment, a current can be applied to the culture between the first working electrode part on one side and the first working electrode part on the other side.

[0024] No. 6 According to the culture device of the embodiment, a current can be applied to the culture between the second working electrode part on one side and the second working electrode part on the other side.

[0025] No. 7 According to the culture device of the embodiment, the current density for the culture between the first working electrode part on one side and the second working electrode part on the other side can be made closer to the current density for the culture between the first working electrode part on one side and the first working electrode part on the other side, thereby making the current density for the culture uniform in the second direction.

[0026] No. 8 According to the embodiment of the culture device, TEER measurement can be performed using the four-terminal method by applying a current between the first working electrode part on one side and the first working electrode part on the other side and measuring the voltage between the first reference electrode part on one side and the first reference electrode part on the other side.

[0027] No. 9 According to the culture device of this embodiment, a measurement chamber with working electrodes arranged on both sides can be easily formed by stacking the first member, the second member, the one-side electrode substrate, the other-side electrode substrate, and the permeable layer in a predetermined order.

[0028] No. 10 In the culture device of the embodiment, the one-side working electrode has an electrode portion that extends from one end to the other end of the measurement chamber in the first direction, so that a current can be applied to the entire area in the longitudinal direction of the measurement chamber. [Brief explanation of the drawings]

[0029] [Figure 1]1A and 1B are a top view and a bottom view, respectively, showing a culture device according to an embodiment. [Figure 2] 2 is a cross-sectional view of the culture device taken along line AA in FIG. 1. FIG. [Figure 3] 2 is a cross-sectional view of the culture device taken along line BB in FIG. 1. FIG. [Figure 4] FIG. 1 is a circuit diagram for measuring the resistance value of a cell. [Figure 5] FIG. 10 is a diagram showing a simulation result of current density using an analytical model according to a comparative example. [Figure 6] FIG. 2 is a diagram showing the results of a simulation of current density using an analytical model corresponding to the culture device shown in FIG. [Figure 7] FIG. 7 is a diagram showing the resistance value of a cell calculated from the results of a current density simulation using the analytical model shown in FIG. 6. [Figure 8] FIG. 10 is a cross-sectional view of a culture device according to a first modified example. [Figure 9] FIG. 10 is a cross-sectional view of a culture device according to a second modified example. DETAILED DESCRIPTION OF THE INVENTION

[0030] Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings. Note that the components described in the embodiment are merely examples and are not intended to limit the scope of the present invention. In the drawings, the length and number of each part may be exaggerated or simplified as necessary to facilitate understanding.

[0031] 1. First embodiment FIG. 1 is a top view (A) and a bottom view (B) showing a culture device 1 according to an embodiment. Note that FIG. 1 shows a measurement chamber 100 and the structure around the measurement chamber 100 in the culture device 1. FIG. 2 is a cross-sectional view of the culture device 1 taken along line AA shown in FIG. 1. FIG. 3 is a cross-sectional view of the culture device 1 taken along line BB shown in FIG. 1.

[0032] In the following description, the terms "first direction," "second direction," and "vertical direction (third direction)" are defined to explain the positional relationship of each element. The second direction intersects with the first direction, and more preferably is perpendicular to the first direction. The vertical direction intersects with the first and second directions, and more preferably is perpendicular to the first and second directions. In the following description, the upper side in the vertical direction may be simply abbreviated as "upper side," and the lower side in the vertical direction may be simply abbreviated as "lower side."

[0033] As shown in FIG. 1, the culture device 1 is a so-called microchannel device in which the internal space, that is, a measurement chamber 100, is a closed space except for a minute supply channel 14 and a discharge channel 15. The culture device 1 is used for TEER measurement, which measures the electrical resistance (resistance, instance, or impedance) of cells 9 (cultured material) cultured in the measurement chamber 100 using a four-terminal method. Note that the culture material to be measured is not limited to cells 9, and may also be a biological sample such as living tissue.

[0034] 2 and 3, the culture device 1 includes a measurement container 10, an upper electrode substrate 21 (one-side electrode substrate), and a lower electrode substrate 22 (other-side electrode substrate). The measurement container 10 is composed of a plate-shaped first member 11 and a second member 12. The second member 12 is disposed below the first member 11. The first member 11 and the second member 12 are formed of, for example, PET (polyethylene terephthalate).

[0035] The measurement container 10 has a measurement chamber 100 therein. The measurement chamber 100 forms a space capable of containing a liquid such as a culture medium. The side surfaces of the measurement chamber 100 are formed by inner surfaces 13a and 13b of through holes formed in the first member 11 and the second member 12. As shown in FIG. 3, the measurement chamber 100 has a first side surface 101 on one side in the second direction and a second side surface 102 on the other side in the second direction. The first side surface 101 and the second side surface 102 face each other in the second direction.

[0036] 1, the length of the measurement chamber 100 in the first direction is longer than the length in the second direction. In this example, the measurement chamber 100 has a rectangular shape in a plan view seen from above, with the first direction as the longitudinal direction and the second direction as the lateral direction.

[0037] The upper electrode substrate 21 and the lower electrode substrate 22 are substrates made of, for example, quartz glass, and are transparent substrates. As shown in FIGS. 2 and 3, the upper electrode substrate 21 is disposed above the first member 11. The lower electrode substrate 22 is disposed below the second member 12. As shown in FIGS. 2 and 3, the lower surface of the upper electrode substrate 21 closes the upper opening of the measurement chamber 100. In addition, the upper surface of the lower electrode substrate 22 closes the lower opening of the measurement chamber 100.

[0038] 1(A), the upper electrode base material 21 is transparent, so that the upper working electrode 40, the upper reference electrode 60, the measurement chamber 100, and the like arranged inside the culture device 1 can be seen from above. Similarly, as shown in FIG. 1(B), the lower electrode base material 22 is transparent, so that the lower working electrode 50, the lower reference electrode 70, the measurement chamber 100, and the like arranged inside the culture device 1 can be seen from below.

[0039] 2 and 3, the culture device 1 has a permeable layer 30. The permeable layer 30 is a sheet-like permeable membrane that allows liquid to permeate. The permeable layer 30 is made of, for example, PC (polycarbonate), PTFE (polytetrafluoroethylene), or PET. The permeable layer 30 is sandwiched and held between a first member 11 and a second member 12.

[0040] The permeable layer 30 is a layer that vertically divides the measurement chamber 100 into an upper first chamber 110 and a lower second chamber 120. The liquid contained in the measurement chamber 100 can move between the first chamber 110 and the second chamber 120 via the permeable layer 30. The first chamber 110 is formed by the inner surface 13a of the first member 11, and the second chamber 120 is formed by the inner surface 13b of the second member 12.

[0041] As shown in FIGS. 1 and 2, the culture device 1 has a supply port 210 and a supply flow path 14. The supply flow path 14 is a flow path through which a liquid supplied from the outside to the measurement chamber 100 passes. The supply port 210 is an opening for supplying a liquid from the outside to the supply flow path 14. In this example, the supply port 210 is configured as a hole that vertically penetrates the upper electrode base material 21. The supply port 210 is in communication with the first chamber 110 of the measurement chamber 100 via the supply flow path 14. Note that "communication" refers to a state in which the two are connected so that a liquid can flow through them. The supply flow path 14 is formed in a tubular shape extending along the first direction. As shown in FIG. 2, the supply flow path 14 is configured by the inner surface of the hole that vertically penetrates the first member 11, the lower surface of the upper electrode base material 21, and the upper surface of the second member 12.

[0042] As shown in FIGS. 1 and 2, the culture device 1 has an outlet 220 and an outlet flow path 15. The outlet flow path 15 is a flow path through which liquid passes to be discharged from the measurement chamber 100 to the outside. The outlet 220 is an opening for discharging liquid from the outlet flow path 15. In this example, the outlet 220 is formed by a hole that vertically penetrates the upper electrode substrate 21 and the first member 11. The outlet 220 is connected to the second chamber 120 of the measurement chamber 100 via the outlet flow path 15. The outlet flow path 15 is formed in a tubular shape extending along the first direction. As shown in FIG. 2, the outlet flow path 15 is formed by the inner surface of the hole that vertically penetrates the second member 12, the lower surface of the first member 11, and the upper surface of the lower electrode substrate 22.

[0043] When measuring the electrical resistance of cells 9 using the culture device 1, a supply tube for supplying a liquid such as culture medium to the measurement chamber 100 is connected to the supply port 210, and a discharge tube for discharging the culture medium from the measurement chamber 100 is connected to the discharge port 220.

[0044] As shown in FIG. 1, the culture device 1 further includes an upper working electrode 40 (one-side working electrode), a lower working electrode 50 (the other-side working electrode), an upper reference electrode 60 (one-side reference electrode), and a lower reference electrode 70 (the other-side reference electrode).

[0045] The upper working electrode 40 and the upper reference electrode 60 are each disposed on the lower surface of the upper electrode substrate 21. The upper working electrode 40 and the upper reference electrode 60 are disposed on the upper side (one side in the third direction) with respect to the measurement chamber 100. The lower working electrode 50 and the lower reference electrode 70 are each disposed on the upper surface of the lower electrode substrate 22. The lower working electrode 50 and the lower reference electrode 70 are disposed on the lower side (the other side in the third direction) with respect to the measurement chamber 100.

[0046] The upper working electrode 40 and the upper reference electrode 60 are formed, for example, by vapor deposition of electrode metal on the lower surface of the upper electrode substrate 21. At least the portions of the upper working electrode 40 and the upper reference electrode 60 formed by vapor deposition that vertically overlap the measurement chamber 100 are preferably covered with an insulating protective film (oxide film) or the like. Covering them with an insulating protective film in this way can suppress electrochemical reactions that occur at the interface between the electrode metal and the liquid, and can also suppress deterioration and wear of the electrode metal over time. Like the upper working electrode 40 and the upper reference electrode 60, the lower working electrode 50 and the lower reference electrode 70 are also formed on the upper surface of the lower electrode substrate 22 by vapor deposition and are appropriately covered with an insulating protective film.

[0047] <Upper working electrode 40> As shown in FIG. 1(A), the upper working electrode 40 has a working electrode portion 41 (one-side first working electrode portion), a working electrode portion 42 (one-side second working electrode portion), and a working bus portion 43. As shown in FIG. 1(A), the working electrode portions 41 and 42 are longer in the first direction than in the second direction. In this example, the working electrode portions 41 and 42 extend linearly along the first direction. As shown in FIG. 1(A), one end of each of the working electrode portions 41 and 42 in the first direction is electrically connected to the working bus portion 43.

[0048] 1(A) and 3, the working electrode unit 42 is disposed apart from the working electrode unit 41 in the second direction. The working electrode unit 41 is disposed at a position closer to one side in the second direction than the center of the measurement chamber 100. The working electrode unit 42 is disposed at a position closer to the other side in the second direction than the center of the measurement chamber 100.

[0049] 3, the working electrode unit 41 is disposed at a distance W31 on the other side in the second direction from the first side surface 101 of the measurement chamber 100. The working electrode unit 42 is disposed at a distance W32 on one side in the second direction from the second side surface 102 of the measurement chamber 100.

[0050] <Lower working electrode 50> As shown in FIG. 1(B), the lower working electrode 50 has a working electrode portion 51 (the other-side first working electrode portion), a working electrode portion 52 (the other-side second working electrode portion), and a working bus portion 53. The working electrode portions 51 and 52 are longer in the first direction than in the second direction. In this example, the working electrode portions 51 and 52 extend linearly along the first direction. One end of each of the working electrode portions 51 and 52 in the first direction is electrically connected to the working bus portion 53.

[0051] 1(B) and 3, the working electrode unit 52 is disposed away from the working electrode unit 51 on the other side in the second direction. The working electrode unit 51 is disposed at a position closer to one side in the second direction with respect to the center of the measurement chamber 100. The working electrode unit 52 is disposed at a position closer to the other side in the second direction with respect to the center of the measurement chamber 100. As shown in FIG. 3, the working electrode unit 52 is disposed away from the working electrode unit 41 on the other side in the second direction.

[0052] 3, the working electrode unit 51 is disposed at a distance W33 on the other side in the second direction from the first side surface 101 of the measurement chamber 100. The working electrode unit 52 is disposed at a distance W34 on one side in the second direction from the second side surface 102 of the measurement chamber 100.

[0053] The widths (dimensions in the second direction) W11, W12, W13, and W14 of the working electrode portions 41, 42, 51, and 52 are preferably the same size.

[0054] As shown in Fig. 3, the working electrode units 41 and 51 are arranged at the same position in the second direction. In the measurement chamber 100, the working electrode unit 41 faces the working electrode unit 51 in the up-down direction. As shown in Fig. 3, the working electrode units 42 and 52 are arranged at the same position in the second direction. In the measurement chamber 100, the working electrode unit 42 faces the working electrode unit 52 in the up-down direction.

[0055] The working electrode sections 41, 42, 51, and 52 are each disposed at a position where they overlap vertically with the measurement chamber 100. As shown in FIGS. 1 and 2, the length of each of the working electrode sections 41, 42, 51, and 52 in the first direction is longer than the length of the measurement chamber 100 in the first direction. As shown in FIGS. 1 and 2, the working electrode sections 41, 42, 51, and 52 are each disposed so as to cross the measurement chamber 100 in the first direction. That is, the upper working electrode 40 and the lower working electrode 50 each have an electrode portion (working electrode section 41, 42, 51, and 52) extending from one end to the other end of the measurement chamber 100 in the first direction.

[0056] <Upper reference electrode 60> As shown in Figures 1(A) and 3, the upper reference electrode 60 has a reference electrode portion 61 (one side first reference electrode portion), a reference electrode portion 62 (one side second reference electrode portion), and a reference bus portion 63.

[0057] 1A, the length of the reference electrode portions 61 and 62 in the first direction is longer than the length in the second direction. In this example, the reference electrode portions 61 and 62 extend linearly along the first direction. One end of each of the reference electrode portions 61 and 62 in the first direction is electrically connected to a reference bus portion 63.

[0058] 3, the reference electrode part 61 is disposed at a position closer to one side in the second direction than the center of the measurement chamber 100. The reference electrode part 62 is disposed at a position closer to the other side in the second direction than the center of the measurement chamber 100.

[0059] 3, the reference electrode portion 62 is disposed on the other side in the second direction relative to the reference electrode portion 61, at a distance W51. The reference electrode portions 61, 62 are disposed between the working electrode portions 41, 42 in the second direction.

[0060] 3, the reference electrode part 61 is arranged at a distance W41 on the other side in the second direction from the working electrode part 41. The reference electrode part 62 is arranged at a distance W42 on one side in the second direction from the working electrode part 42.

[0061] In the culture device 1, a gap is formed between the working electrode units 41, 42 over the entire longitudinal direction (first direction) of the measurement chamber 100. The reference electrode units 61, 62 of the upper reference electrode 60 are disposed in this gap. A gap of a distance W51 is also formed between the reference electrode units 61, 62. Therefore, an observer can observe the cells 9 supported on the permeable layer 30 from above the culture device 1 through the gap between the reference electrode units 61, 62.

[0062] The widths (dimensions in the second direction) W21, W22 of the reference electrode portions 61, 62 of the upper reference electrode 60 are preferably smaller than the widths W11, W12 of the working electrode portions 41, 42 of the upper working electrode 40. By reducing the widths W21, W22 of the reference electrode portions 61, 62 in this manner, it is possible to prevent the upper reference electrode 60 from interfering with the observation of the cell 9.

[0063] <Lower reference electrode 70> The lower reference electrode 70 has a reference electrode portion 71 (other-side first reference electrode portion), a reference electrode portion 72 (other-side second reference electrode portion), and a reference bus portion 73. The length of the reference electrode portion 71 and the reference electrode portion 72 in the first direction is longer than the length in the second direction. In this example, the reference electrode portion 71 and the reference electrode portion 72 extend linearly along the first direction. One end of each of the reference electrode portion 71 and the reference electrode portion 72 in the first direction is electrically connected to the reference bus portion 73.

[0064] 1, the working bus sections 43 and 53 and the reference bus sections 63 and 73 each have a contact section for electrically connecting to an external device, which is exposed to the outside of the culture device 1 so that an electrode (such as a probe pin) of the external device can contact the contact section.

[0065] 3, the reference electrode part 71 is disposed at a position closer to one side in the second direction than the center of the measurement chamber 100. The reference electrode part 72 is disposed at a position closer to the other side in the second direction than the center of the measurement chamber 100.

[0066] 3, the reference electrode portion 72 is disposed on the other side in the second direction relative to the reference electrode portion 71, separated by a distance W52. The reference electrode portions 71 and 72 are disposed between the working electrode portions 51 and 52 in the second direction.

[0067] As shown in Fig. 3, the reference electrode units 61 and 71 are arranged at the same position in the second direction. In the measurement chamber 100, the reference electrode unit 61 faces the reference electrode unit 71 in the vertical direction. Also, as shown in Fig. 3, the reference electrode units 62 and 72 are arranged at the same position in the second direction. In the measurement chamber 100, the reference electrode unit 62 faces the reference electrode unit 71 in the vertical direction.

[0068] The widths W21, W22, W23, and W24 of the reference electrode portions 61, 62, 71, and 72 are preferably the same. The widths W23 and W24 of the reference electrode portions 71 and 72 are preferably smaller than the widths W13 and W14 of the working electrode portions 51 and 52 of the lower working electrode 50.

[0069] In the measurement chamber 100, the reference electrode portion 61 overlaps with the reference electrode portion 71, and the reference electrode portion 62 overlaps with the reference electrode portion 72 in the vertical direction. The lengths of the reference electrode portions 61, 62, 71, and 72 in the first direction are longer than the length of the measurement chamber 100 in the first direction. As shown in FIGS. 1(A) and 1(B), the reference electrode portions 61, 62, 71, and 72 are arranged so as to cross the measurement chamber 100 in the first direction. That is, the upper reference electrode 60 and the lower reference electrode 70 have electrode portions (reference electrode portions 61, 62, 71, and 72) that extend from one end to the other end of the measurement chamber 100 in the first direction.

[0070] The length of the measurement chamber 100 in the first direction is preferably 100 mm or less, more preferably 20 mm or more and 30 mm or less. The length of the measurement chamber 100 in the second direction is preferably 10 mm or less, more preferably 1 mm or more and 2 mm or less. The length of the measurement chamber 100 in the vertical direction is preferably 10 mm or less, more preferably 1 mm or more and 2 mm or less.

[0071] The widths W11 to W14 of the working electrode portions 41, 42, 51, and 52 are preferably 1 mm or less, more preferably 300 μm to 500 μm, and are illustratively 400 μm. The widths W11 to W14 are preferably the same, but may be different.

[0072] 3, the widths W21 to W24 of the reference electrode portions 61, 62, 71, and 72 are preferably 200 μm or less, more preferably 50 μm to 150 μm, and are illustratively 100 μm. Note that the widths W21 to W24 are preferably the same, but may be different.

[0073] 3, the distances W31, W32, W33, and W34 between the working electrode unit 41 and the first side surface 101, between the working electrode unit 42 and the second side surface 102, between the working electrode unit 51 and the first side surface 101, and between the working electrode unit 52 and the second side surface 102 are preferably 100 μm or more and 300 μm or less, and are illustratively 200 μm. Note that the distances W31 to W34 are preferably the same, but may be different.

[0074] 3, the distances W41, W42, W43, and W44 between the working electrode part 41 and the reference electrode part 61, between the working electrode part 42 and the reference electrode part 62, between the working electrode part 51 and the reference electrode part 71, and between the working electrode part 52 and the reference electrode part 72 are preferably 200 μm or less, and are illustratively 100 μm. The distances W41 to W44 are preferably the same, but may be different.

[0075] 3, the distances W51 and W52 between the reference electrode portions 61 and 62 and between the reference electrode portions 71 and 72 are preferably 300 μm or more and 500 μm or less, and are illustratively 400 μm. The distances W51 and W52 are preferably the same, but may be different.

[0076] In a plan view seen from above, the area of ​​the working electrode portions 41, 42 of the upper working electrode 40 that overlap with the measurement chamber 100 is preferably 30% or more and 50% or less of the area of ​​the measurement chamber 100, and is illustratively 40%.

[0077] <About measuring electrical resistance> 4 is a circuit diagram for measuring the electrical resistance of cell 9. When measuring the electrical resistance of cell 9, a power supply 91 and a voltmeter 92 are connected to the culture device 1. The output terminal of the power supply 91 is electrically connected to the contact portion of the upper working electrode 40 and the contact portion of the lower working electrode 50 via conductor 94a. The input terminal of the voltmeter 92 is electrically connected to the contact portion of the upper reference electrode 60 and the contact portion of the lower reference electrode 70 via conductor 94b.

[0078] When measuring the electrical resistance of cells 9, a large number of cells 9 are supported on the upper surface of the permeable layer 30 in the measurement chamber 100. Then, a liquid such as a culture medium is filled into the measurement chamber 100 via the supply port 210 and the supply flow path 14. The liquid in the measurement chamber 100 is discharged via the discharge port 220 and the discharge flow path 15. This causes the exchange (or circulation) of the liquid between the first chamber 110 and the second chamber 120 of the measurement chamber 100.

[0079] 4, resistance Rm corresponds to the electrical resistance of the portion of permeation layer 30 located in measurement chamber 100 and the layer of cells 9 (hereinafter referred to as "cell layer 90") supported by that portion of permeation layer 30. Resistance Rw1 corresponds to the electrical resistance of the liquid between upper working electrode 40 and cell layer 90 (i.e., first chamber 110). Resistance Rw2 corresponds to the electrical resistance of the liquid between lower working electrode 50 and cell layer 90 (i.e., second chamber 120).

[0080] 4, resistance Rr1 corresponds to the electrical resistance of the liquid between the upper reference electrode 60 and the cell layer 90 (i.e., the first chamber 110). Resistance Rr2 corresponds to the electrical resistance of the liquid between the lower reference electrode 70 and the cell layer 90 (i.e., the second chamber 120).

[0081] A current is applied between the upper working electrode 40 and the lower working electrode 50 by the power supply device 91, and the voltage between the upper reference electrode 60 and the lower reference electrode 70 is measured by the voltmeter 92. Then, the electrical resistance between the upper working electrode 40 and the lower working electrode 50 is calculated from the voltage value measured by the voltmeter 92. Furthermore, the resistance Rm of the cell layer 90 is calculated from the calculated electrical resistance between the upper working electrode 40 and the lower working electrode 50.

[0082] When the power supply 91 applies a current between the upper working electrode 40 and the lower working electrode 50, oxidation and reduction reactions of the liquid may occur on the surfaces of the upper working electrode 40 and the lower working electrode 50, forming an electric double layer. In this case, the output voltage from the power supply 91 may differ from the voltage applied between the upper working electrode 40 and the lower working electrode 50. In the case of the culture device 1, an upper reference electrode 60 and a lower reference electrode 70 are disposed near the upper working electrode 40 and the lower working electrode 50, respectively, inside the measurement chamber 100. Therefore, the voltage between the upper reference electrode 60 and the lower reference electrode 70 is measured, and the measured voltage is used as the voltage between the upper working electrode 40 and the lower working electrode 50, thereby enabling accurate measurement of the resistance Rm of the cell layer 90.

[0083] <Effects> According to the culture device 1, by extending the upper working electrode 40 and the lower working electrode 50 in a first direction, which is the longitudinal direction of the measurement chamber 100, the length of the electrode portion facing the measurement chamber 100 can be made longer than when the electrodes are extended in the lateral direction of the measurement chamber 100. This makes it possible to widen the area to which current is applied without increasing the number of electrodes or making the electrodes thicker. Therefore, current can be effectively applied to a wide area of ​​the measurement chamber 100 while preventing the upper working electrode 40 from interfering with observation of the cells 9.

[0084] The upper working electrode 40 has electrode portions (working electrode portions 41, 42) extending from one end to the other end of the measurement chamber 100 in the first direction. This allows current to be applied to the entire longitudinal area of ​​the measurement chamber 100, thereby allowing current to be applied to a wide area of ​​the measurement chamber 100. The lower working electrode 50 also has electrode portions (working electrode portions 51, 52) extending from one end to the other end of the measurement chamber 100 in the first direction. This allows current to be applied to the entire longitudinal area of ​​the measurement chamber 100.

[0085] Since the working electrode sections 41 and 51 face each other, a current can be applied to the cell 9 located between the working electrode sections 41 and 51. Furthermore, since the working electrode sections 42 and 52 face each other, a current can be applied to the cell 9 located between the working electrode sections 42 and 52.

[0086] The working electrode unit 52 is disposed away from the working electrode unit 41 in the second direction. As a result, a current flows between the working electrode units 41 and 52, thereby applying a current to the intermediate portion of the cell layer 90. Similarly, a current flows between the working electrode units 42 and 51, thereby applying a current to the intermediate portion of the cell layer 90.

[0087] The culture device 1 is composed of a first member 11, a second member 12, an upper electrode substrate 21, a lower electrode substrate 22, and a permeable layer 30. Therefore, by stacking these members one above the other in a predetermined order, it is possible to easily form a measurement chamber 100 in which a pair of upper and lower working electrodes 40 and 50 are arranged on both sides.

[0088] <Simulation> FIG. 5 shows the results of a current density simulation using analytical model 8a according to a comparative example. FIG. 6 shows the results of a current density simulation using analytical model 8b corresponding to the culture device 1 shown in FIG. 1. Specifically, the current density simulation was performed using a finite element method using analytical software such as COMSOL Multiphysics (manufactured by COMSOL AB). In analytical models 8a and 8b, the length of measurement chamber 100 in the first direction (longitudinal direction) is 30 mm, the length in the second direction (transverse direction) is 1 mm, and the length in the vertical direction is 1 mm. In FIGS. 5 and 6, the electrical resistance of cell layer 90 is uniform throughout. To simplify the simulation, analytical models 8a and 8b only include upper working electrode 40 and lower working electrode 50, and upper reference electrode 60 and lower reference electrode 70 are omitted.

[0089] 5(A) is a top view showing the analytical model 8a, and FIG. 5(B) is a side view showing the analytical model 8a. FIG. 5(C) is a diagram showing the simulation results of the current density. In FIG. 5(C), the horizontal axis indicates the position (mm) in the first direction of the measurement chamber 100, and the vertical axis indicates the current density (A / cm 2 ) is shown.

[0090] 5(A) and 5(B), in the analytical model 8a according to the comparative example, the upper working electrode 40 and the lower working electrode 50 are each configured with electrode portions (working electrode portions 41a, 42a, 51a, 52a) whose lengths in the second direction are longer than their lengths in the first direction, and the electrode portions are disposed at both ends in the first direction of the measurement chamber 100. The working electrode portions 41a, 51a and the working electrode portions 42a, 52a face each other vertically.

[0091] In the case of the analytical model 8a, as shown in Fig. 5(C), the current density decreases as it approaches the center of the measurement chamber 100 in the first direction. Specifically, the current density directly below the electrode is approximately 1.00 × 10 -9 whereas the current density near the center of the measurement chamber 100 is approximately 1.00 × 10 -12 There is a difference of about 1000 times between the two. In other words, in the case of an electrode shape like analytical model 8a, large variations in current density occur.

[0092] 6(A) is a top view showing the analytical model 8b, and FIG. 6(B) is a cross-sectional view showing the analytical model 8b at a position along the line CC shown in FIG. 6(A). FIGS. 6(C) and 6(D) are diagrams showing the simulation results of the current density. In FIG. 6(C), the horizontal axis indicates the position (mm) in the first direction of the measurement chamber 100, and the vertical axis indicates the current density (A / cm 2 6(D), the horizontal axis indicates the position (mm) in the second direction of the measurement chamber 100, and the vertical axis indicates the current density (A / cm 2 ) is shown.

[0093] In the case of analytical model 8b, as shown in Figure 6(C), there is almost no variation in current density in the first direction. Also, in the case of analytical model 8b, as shown in Figure 6(D), the difference in current density in the second direction is less than 5%. These simulation results show that with the electrode shape of analytical model 8b, current can be applied uniformly to the entire cell 9.

[0094] In the case of the analytical model 8b, as shown in FIG. 6(B), the distance L2 (second distance) between the working electrode portions 41 and 52 is sufficiently small compared to the distance L1 (first distance, 1 mm in this case) between the working electrode portions 41 and 51. Therefore, a sufficient current flows between the working electrode portions 41 and 52. Similarly to the current flowing between the working electrode portions 41 and 52, a sufficient current also flows between the working electrode portions 42 and 51. Therefore, as shown in FIG. 6(D), it is considered that the current density is uniform in the second direction.

[0095] It is desirable that the distance L2 be 1.5 times the distance L1 or less. If the distance L2 is greater than 1.5 times the distance L1, the working electrode unit 52 moves away from the working electrode unit 41 in the second direction. This causes the current density in the cells 9 between the working electrode units 41 and 52 to be significantly smaller than the current density in the cells 9 between the working electrode units 41 and 51. This may result in variations in the current density in the cell layer 90.

[0096] Fig. 7 is a diagram showing the resistance value of the cell layer 90 calculated from a simulation using the analytical model 8b shown in Fig. 6. Fig. 7 shows the resistance value calculated when the simulation is performed assuming that the resistance of the cell layer 90 is uniform throughout, and the resistance value calculated when the resistance of the cell layer 90 in an area A1 at one end in the first direction or the resistance of the area A2 in the center in the first direction is set to 1 / 10 or 1 / 100 of the resistance of the other areas.

[0097] As shown in Figure 7, when the electrical resistance of the end region A1 and the resistance of the central region A2 are set to 1 / 10, respectively, the calculated resistance values ​​are "197.5Ω" and "197.9Ω." Furthermore, when the resistance of the end region A1 and the resistance of the central region A2 are set to 1 / 100, respectively, the calculated resistance values ​​are "190.6Ω" and "191.3Ω." Thus, in the case of analysis model 8b, the resistance value of cell layer 90 calculated from the current density remains approximately the same regardless of whether the resistance value of either the end region A1 or the central region A2 in cell layer 90 is varied.

[0098] In the case of analytical model 8a, as shown in FIG. 5(C), the current density varies between the end and center of the cell layer 90. Therefore, the resistance value of the cell layer 90 calculated from the current density may differ between a case where the resistance of an end region A1 of the cell layer 90 is low and a case where the resistance of a central region A2 of the cell layer 90 is low. In contrast, in the case of analytical model 8b, as shown in FIG. 6(C), the current density is uniform in the first direction. Therefore, even if a portion of the cell layer 90 has low resistance, the current flows uniformly to other portions. Therefore, regardless of the location of a portion of the cell layer 90 where the resistance differs from the surrounding area, the current can be applied uniformly to other portions of the cell layer 90, and ultimately, a similar resistance value can be calculated. Therefore, the electrode shape of analytical model 8b improves the reproducibility of TEER measurements compared to the electrode shape of analytical model 8a.

[0099] As described above, the analysis results of the analytical model 8b show that by extending the working electrode sections 41, 42, 51, and 52 in the longitudinal direction of the measurement chamber 100, a current can be applied uniformly to a wide area of ​​the cell layer 90. Furthermore, even if the resistance of a portion of the cell layer 90 fluctuates due to death or absence of cells 9, a current flows uniformly in other portions of the cell layer 90. Therefore, the resistance of the cell layer 90 can be calculated appropriately.

[0100] <2. Modifications> Although the embodiments have been described above, the present invention is not limited to the above and various modifications are possible.

[0101] FIG. 8 is a cross-sectional view of the culture device 1 according to a first modified example. As shown in FIG. 8, the working electrode portions 41 and 42 of the upper working electrode 40 may extend to the outside of the measurement chamber 100 in the second direction. In this first modified example, the width W11 of the working electrode portion 41 is, for example, 800 μm. By extending the working electrode portions 41 and 42 to the outside of the measurement chamber 100 in this manner, a current can be applied to the end of the measurement chamber 100 in the second direction. Furthermore, as shown in FIG. 8, the working electrode portions 51 and 52 of the lower working electrode 50 may extend to the outside of the measurement chamber 100 in the second direction.

[0102] Fig. 9 is a cross-sectional view of a culture device 1 according to a second modification. As shown in Fig. 9, the upper working electrode 40 may further include working electrode portions 44 and 45 in addition to working electrode portions 41 and 42. The working electrode portions 44 and 45 extend linearly along the first direction.

[0103] The reference electrode portion 61 is disposed between the working electrode portions 41 and 44 in the second direction. The reference electrode portion 62 is disposed between the working electrode portions 42 and 45 in the second direction. In this second modified example, the width W11 of the working electrode portion 41 is, for example, 500 μm. The width W15 of the working electrode portion 44 is, for example, 200 μm. The distance W45 between the reference electrode portion 61 and the working electrode portion 44 is, for example, 100 μm. The distance W53 between the working electrode portions 44 and 45 is, for example, 400 μm.

[0104] 9, the lower working electrode 50 may further include working electrode portions 54 and 55 in addition to the working electrode portions 51 and 52. The working electrode portions 54 and 55 extend linearly along the first direction. The reference electrode portion 71 may be disposed between the working electrode portions 51 and 54 in the second direction. Furthermore, the reference electrode portion 72 may be disposed between the working electrode portions 52 and 55 in the second direction.

[0105] Although the present invention has been described in detail, the above description is merely illustrative in all respects and does not limit the present invention. It is understood that countless variations not illustrated can be envisioned without departing from the scope of the present invention. The configurations described in the above embodiments and variations can be combined or omitted as appropriate as long as they are not mutually inconsistent. [Explanation of symbols]

[0106] 1. Culture device 9 cells 11 First member 12 Second member 13a, 13b inner surface 21 Upper electrode base material (one side electrode base material) 22 Lower electrode base material (other side electrode base material) 30 Transparent layer 40 Upper working electrode (one side working electrode) 41 Working electrode section (first working electrode section on one side) 42 Working electrode part (one side second working electrode part) 50 Lower working electrode (other side working electrode) 51 Working electrode section (first working electrode on the other side) 52 Working electrode section (second working electrode on the other side) 60 Upper reference electrode (one-side reference electrode) 70 Lower reference electrode (other side reference electrode) 90 cell layers 100 measurement room 110 Room 1 120 Room 2

Claims

1. A culture device applicable to measuring the electrical resistance of a culture, comprising: a measurement chamber having a length in a first direction longer than a length in a second direction intersecting the first direction; a permeable layer that partitions the measurement chamber into a first chamber on one side and a second chamber on the other side in a third direction that intersects the first direction and the second direction, and that allows a liquid to pass through; a one-side working electrode located on one side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction; a second working electrode located on the second side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction; a one-side reference electrode located on one side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction; Equipped with The one side working electrode is a first working electrode portion extending in the first direction; a one-side second working electrode portion extending in the first direction and spaced apart from the one-side first working electrode portion in the second direction; an action bus portion to which one ends of the one-side first working electrode portion and the one-side second working electrode portion are connected; and the one-side reference electrode has a one-side reference electrode portion located between the one-side first working electrode portion and the one-side second working electrode portion, the width of the one-side reference electrode portion is smaller than the widths of the one-side first working electrode portion and the one-side second working electrode portion; a culture device, wherein the one-side first working electrode portion and the one-side second working electrode portion extend to the outside of the measurement chamber in the second direction;

2. 10. The culture device of claim 1, The other-side working electrode extends in the first direction and has a second-side first working electrode portion facing the one-side working electrode in the measurement chamber.

3. The culture device according to claim 1 or claim 2, The other-side working electrode extends in the first direction and has an other-side second working electrode portion located in the measurement chamber away from the one-side working electrode in the second direction.

4. The culture device of claim 3, The other working electrode is a second working electrode portion extending in the first direction; an other-side second working electrode portion extending in the first direction and spaced apart from the other-side first working electrode portion in the second direction; A culture device comprising:

5. The culture device of claim 4, A culture device, wherein the one-side first working electrode unit faces the other-side first working electrode unit in the measurement chamber.

6. 6. The culture device of claim 5, A culture device, wherein the one-side second working electrode portion faces the other-side second working electrode portion in the measurement chamber.

7. The culture device according to any one of claims 4 to 6, a culture device in which, when a distance between the one-side first working electrode portion and the other-side first working electrode portion is defined as a first distance and a distance between the one-side first working electrode portion and the other-side second working electrode portion is defined as a second distance, the second distance is 1.5 times or less of the first distance.

8. The culture device according to any one of claims 1 to 7, an other-side reference electrode located on the other side of the measurement chamber in the third direction and having an electrode portion whose length in the first direction is longer than its length in the second direction; The culture device further comprises:

9. The culture device according to any one of claims 1 to 8, a first member having an inner surface that defines the first chamber; a second member located on the other side of the first member in the third direction and having an inner surface that forms the second chamber; a first electrode substrate located on one side of the first member in the third direction and having a surface on which the first working electrode is disposed; a second electrode substrate located on the second side in the third direction relative to the second member and having a surface on which the second working electrode is disposed; Furthermore, The culture device, wherein the permeable layer is disposed between the first member and the second member.

10. The culture device according to any one of claims 1 to 9, the one-side working electrode has an electrode portion extending from one end to the other end of the measurement chamber in the first direction, a culture device, wherein the other working electrode has an electrode portion extending from one end to the other end of the measurement chamber in the first direction;

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