JP7730669B2 - Culture device and method for manufacturing same - Google Patents

Description

The subject matter disclosed herein relates to a culture device and a method for manufacturing the same.

Techniques for measuring the electrical resistance of cultured cells are known to investigate the properties and culture state of the cells. 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 the cells cultured on the membrane. Such techniques for measuring the electrical resistance of cells are described, for example, in Patent Document 1.

Patent Document 2 also describes a method in which electrodes are provided on the lid of a culture vessel, and electrodes are provided above and below the cell culture section within a cell culture channel device, allowing measurements to be performed while maintaining the culture environment.

Japanese Patent Application Laid-Open No. 2005-137307 Special table 2017-513483 publication

However, in the case of Patent Document 2, electrodes are placed above and below the lid and cell culture section. This requires the electrodes to be formed in separate components, making it difficult to reduce the cost of forming the electrodes.

The objective of the present invention is to provide technology that can reduce the cost of forming electrodes.

In order to solve the above problem, a first aspect is an incubation device comprising: a measurement container; and an electrode base material arranged on one side of the measurement container in a first direction, wherein the measurement container has a measurement chamber capable of containing a liquid and a measurement flow path connected to the measurement chamber at one end and extending from the measurement chamber to the one side in the first direction, wherein the electrode base material has a first surface facing the other side in the first direction, a first electrode arranged on the first surface and facing into the measurement chamber, and a second electrode arranged on the first surface and facing into the measurement flow path, wherein the measurement container further comprises a permeable layer that is permeable to a liquid and divides the measurement chamber into a first chamber on one side in the first direction and a second chamber on the other side in the first direction, wherein the measurement container has a first member that forms the first chamber and a second member that forms the second chamber, and the permeable layer is arranged between the first member and the second member in the first direction. The culture device further has a conductive portion electrically connected to the first electrode or the second electrode, and the conductive portion is arranged outside the electrode substrate in a second direction intersecting the first direction.

A second aspect is the culture device of the first aspect, in which the first surface of the electrode substrate blocks the opening on one side of the measurement chamber in the first direction and the opening on one side of the measurement flow path in the first direction.

A third aspect is a culture device according to the first or second aspect, wherein the measurement container has a supply flow path connected to the measurement chamber for supplying liquid to the measurement chamber, and a discharge flow path connected to the measurement chamber for discharging liquid from the measurement chamber.

A fourth aspect is the culture device of the third aspect, wherein one end of the discharge flow path is connected to the middle of the measurement flow path.

A fifth aspect is a culture device according to any one of the first to fourth aspects, which is placed in a measurement container and has a conductive hole into which a probe pin can be inserted, and at least one of the first electrode or the second electrode faces the conductive hole.

A sixth aspect is a method for manufacturing a culture device according to any one of the first to fifth aspects, comprising: (a) a step of forming the first electrode and the second electrode on the first surface of the electrode substrate; and (b) a step of attaching the electrode substrate on which the first electrode and the second electrode have been formed by step (a) to one side of the measurement container in the first direction.

According to the culture device of the first aspect, the first electrode and the second electrode are provided on the same electrode substrate, and therefore the cost of forming the electrodes can be reduced compared to when the first electrode and the second electrode are provided in a dispersed manner on multiple components.
Furthermore, cells can be held in the permeable layer while allowing liquid to pass between the top and bottom of the layer, allowing the electrical resistance of the cells to be measured.
Furthermore, by stacking the second member, the transmission layer, and the first member in this order, a measurement chamber partitioned by the transmission layer can be easily formed in the measurement container.
Furthermore, since the conductive portion of the first electrode or the second electrode is disposed on the outside of the electrode base material, the first electrode or the second electrode can be easily electrically connected to an external device.

In the culture device of the second aspect, the openings on one side of the measurement chamber and the measurement flow path can be blocked with an electrode base material, while the first electrode faces the measurement chamber and the second electrode faces the measurement flow path.

According to the culture device of the third aspect, the liquid in the measurement chamber can be replaced.

According to the culture device of the fourth aspect, the measurement flow path and the discharge flow path are partially shared, which allows the measurement container to be made smaller than when the measurement flow path and the discharge flow path are provided independently.

According to the culture device of the fifth aspect, the first electrode or the second electrode can be electrically connected to a measuring device by inserting a probe pin into the conductive hole.

FIG. 2 is an exploded cross-sectional view of the culture device according to the first embodiment. FIG. 2 is a cross-sectional view of the culture device according to the first embodiment. FIG. 1 is a circuit diagram for measuring the electrical resistance of a cell. FIG. 10 is a cross-sectional view of a culture device according to a second embodiment.

Embodiments of the present invention will now be described with reference to the accompanying drawings. Note that the components described in these embodiments are merely examples and are not intended to limit the scope of the present invention. For ease of understanding, the dimensions and number of parts may be exaggerated or simplified as necessary in the drawings.

1. First embodiment
FIG. 1 is an exploded cross-sectional view of a culture device 1 according to a first embodiment. FIG. 2 is a cross-sectional view of the culture device 1 according to the first embodiment. The culture device 1 is a so-called microchannel device in which a measurement chamber 100, which is an internal space, is a closed space except for a minute supply channel 51 and a discharge channel 53. The culture device 1 is an apparatus that measures the electrical resistance (resistance, instance, or impedance) of cells 9 (or a biological sample such as tissue) cultured in the measurement chamber 100 using a four-terminal measurement method. As shown in FIGS. 1 and 2 , the culture device 1 includes a measurement container 10 and an electrode substrate 20.

In the following description, the direction in which the measurement container 10 and electrode substrate 20 are aligned in the culture device 1 is referred to as the "vertical direction" (first direction). The direction from the measurement container 10 toward the electrode substrate 20 is referred to as the "upward direction" (one side of the first direction). The direction from the electrode substrate 20 toward the measurement container 10 is referred to as the "downward direction" (the other side of the first direction).

As shown in Figure 2, the measurement container 10 has a measurement chamber 100 inside. The measurement chamber 100 forms a space capable of containing a liquid such as a culture medium. As shown in Figures 1 and 2, the measurement container 10 of this embodiment has plate-shaped first member 11, second member 12, third member 13, and fourth member 14. A single measurement container 10 is formed by stacking the third member 13 above the fourth member 14, the second member 12 above the third member 13, and the first member 11 above the second member 12.

The first member 11 to the fourth member 14 are made of, for example, PET (polyethylene terephthalate). To enable observation of the inside of the measurement chamber 100 from above, the first member 11 is preferably translucent, and more preferably colorless and transparent.

As shown in Figure 2, the inner surface of the measurement chamber 100 is formed by the inner surface of a through hole that vertically penetrates the first member 11, the second member 12, and the third member 13. The bottom surface of the measurement chamber 100 is formed by the upper surface of the fourth member 14.

As shown in Figures 1 and 2, the measurement container 10 has a permeable layer 30. The permeable layer 30 is composed of a permeable membrane that allows liquid to pass through. The permeable layer 30 is composed of, for example, PC (polycarbonate), PTFE (polytetrafluoroethylene), or PET. The permeable layer 30 is preferably light-transmitting. The surface of the portion of the permeable layer 30 that is placed within the measurement chamber 100 may be coated with a cell adhesion factor such as collagen. The permeable layer 30 is disposed between the first member 11 and the second member 12. The permeable layer 30 divides the measurement chamber 100 into an upper first chamber 101 and a lower second chamber 102. As shown in Figure 1, a cell 9 is supported on the upper surface of the permeable layer 30. This allows the cell 9 to be placed within the first chamber 101 of the measurement chamber 100.

As shown in FIG. 2, the first chamber 101 of the measurement chamber 100 is formed by the first member 11. More specifically, the first chamber 101 is formed by the inner surface of a through-hole that passes vertically through the first member 11, the upper surface of the transmission layer 30, and the lower surface 21 (first surface) of the electrode substrate 20.

As shown in FIG. 2, the second chamber 102 of the measurement chamber 100 is formed by the second member 12. More specifically, the second chamber 102 is formed by the inner surface of the through-hole that vertically penetrates the second member 12 and the third member 13, the upper surface of the fourth member 14, and the lower surface of the transmission layer 30.

As shown in FIG. 2, the measurement container 10 has a measurement flow path 40 therein. One end of the measurement flow path 40 is connected to the measurement chamber 100. Liquid in the measurement chamber 100 can move to the measurement flow path 40. Also, as shown in FIG. 2, the measurement flow path 40 extends upward from the measurement chamber 100. More specifically, the measurement flow path 40 has a portion that extends in a width direction perpendicular to the up-down direction from the measurement chamber 100, and a portion that extends upward from one end in the width direction. The measurement flow path 40 has a tubular shape surrounded by the inner surfaces of the through-holes that vertically penetrate the first member 11, the permeation layer 30, and the second member 12, and the upper surface of the third member 13.

By constructing the measurement container 10 from the first member 11 to the fourth member 14 stacked on top of each other, the measurement chamber 100, measurement flow path 40, supply flow path 51, and discharge flow path 53 can be easily formed within the measurement container 10. Furthermore, by stacking the second member 12, permeation layer 30, and first member 11 in this order, the measurement chamber 100 separated into two chambers by the permeation layer 30 can be easily formed within the measurement container 10.

The electrode substrate 20 is placed on top of the first member 11 of the measurement container 10. The electrode substrate 20 is a transparent substrate made of, for example, quartz glass. As shown in FIG. 2, the lower surface 21 of the electrode substrate 20 facing downward (i.e., toward the measurement container 10) blocks the upper opening of the measurement chamber 100 and also blocks the upper opening of the measurement flow path 40.

The electrode substrate 20 has working electrodes 61a, 61b and reference electrodes 63a, 63b on its lower surface 21. As shown in FIG. 2, the working electrode 61a and reference electrode 63a face the first chamber 101 of the measurement chamber 100. The working electrode 61b and reference electrode 63b face the measurement flow path 40. The working electrode 61a or the reference electrode 63a is an example of a first electrode. The working electrode 61b or the reference electrode 63b is an example of a second electrode.

As shown in FIG. 2, the working electrode 61a and the reference electrode 63a each have a pair of bifurcated interior electrode portions 611, 631 within the measurement chamber 100. The pair of interior electrode portions 631 of the reference electrode 63a are arranged inside the pair of interior electrode portions 611 of the working electrode 61a in the width direction. The pair of interior electrode portions 631 are arranged with a predetermined gap between them in the width direction. An observer can observe the cells 9 supported on the permeable layer 30 from above the culture device 1 through the gap between the pair of interior electrode portions 631.

The working electrodes 61a, 61b and reference electrodes 63a, 63b are formed, for example, by vapor-depositing electrode metal on the underside 21 of the electrode substrate 20. At least the portions of the vapor-deposited working electrode 61a and reference electrode 63a that vertically overlap the measurement chamber 100 are covered with an insulating protective film (e.g., an oxide film). At least the portions of the working electrode 61b and reference electrode 63b that vertically overlap the measurement flow path 40 are also covered with an insulating protective film. Covering each electrode with an insulating protective film in this manner suppresses electrochemical reactions that occur at the interface between the electrode metal and the liquid, thereby preventing deterioration and wear of the electrode metal over time. The electrode substrate 20 on which the working electrodes 61a, 61b and reference electrodes 63a, 63b are formed is attached to the upper side of the measurement container 10. Furthermore, a top member 70 (described below) is attached to the upper surface of the electrode substrate 20, thereby forming the culture device 1.

The culture device 1 has a top member 70. The top member 70 is arranged above the electrode substrate 20. The top member 70 is made of, for example, PET. The top member 70 has an observation opening 71. The observation opening 71 is a through-hole that passes through the top member 70 from top to bottom. The observation opening 71 is arranged directly above the measurement chamber 100.

The measurement container 10 has a supply flow path 51. The supply flow path 51 is connected to the measurement chamber 100. More specifically, the supply flow path 51 is connected to the first chamber 101 of the measurement chamber 100. The supply flow path 51 forms a flow path for supplying a liquid such as a culture medium to the measurement chamber 100. The supply flow path 51 has a tubular shape surrounded by the inner surface of a through-hole formed in the first member 11, the upper surface of the second member 12, and the lower surface of the electrode substrate 20.

The measurement container 10 has a discharge flow path 53. The discharge flow path 53 is connected to the measurement chamber 100. More specifically, the discharge flow path 53 is connected to the second chamber 102 of the measurement chamber 100. It forms a flow path for discharging a liquid such as a culture medium from the measurement chamber 100 to the outside. The discharge flow path 53 has a tubular shape surrounded by the inner surface of a through-hole formed in the first member 11. In this embodiment, one end of the discharge flow path 53 is connected midway through the measurement flow path 40. That is, the discharge flow path 53 is connected to the second chamber 102 of the measurement chamber 100 via the measurement flow path 40. Note that one end of the discharge flow path 53 may be directly connected to the measurement chamber 100 (preferably the second chamber 102 of the measurement chamber 100) instead of being connected to the measurement flow path 40.

As shown in FIG. 2, the upper end of the supply flow path 51 is connected to a through-hole (supply port 73) that vertically penetrates the top member 70 and the electrode substrate 20. The upper end of the discharge flow path 53 is connected to a through-hole (discharge port 75) that vertically penetrates the top member 70, the electrode substrate 20, and the permeable layer 30.

As shown in FIG. 2, the culture device 1 has conduction holes 81a and 81b. The conduction holes 81a and 81b are formed by the inner surface of a through-hole that passes vertically through the measurement container 10 and the underside 21 of the electrode substrate 20 that covers the upper side of the through-hole. Conductive probe pins 90a and 90b can be inserted into the conduction holes 81a and 81b, respectively. In the illustrated example, the conduction hole 81b is positioned apart from the measurement chamber 100 on one side in the width direction, and the conduction hole 81a is positioned apart from the measurement chamber 100 on the other side in the width direction.

As shown in FIG. 2, the working electrode 61a faces the inside of the conductive hole 81a. The working electrode 61b faces the inside of the conductive hole 81b. When the probe pin 90a inserted into the conductive hole 81a comes into contact with the working electrode 61a, the working electrode 61a is electrically connected to an external device via the probe pin 90a. Furthermore, when the probe pin 90b inserted into the conductive hole 81b comes into contact with the working electrode 61b, the working electrode 61b is electrically connected to an external device via the probe pin 90b. Although not shown, the culture device 1 may also have conductive holes corresponding to the reference electrodes 63a and 63b.

<Measurement of electrical resistance>
3 is a circuit diagram for measuring the electrical resistance of the cell 9. When measuring the electrical resistance of the 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 working electrodes 61a and 61b via conductors 94a. The input terminal of the voltmeter 92 is electrically connected to the reference electrodes 63a and 63b via conductors 94b. The connection between the conductor 94a and the working electrodes 61a and 61b may be made via the probe pins 90a and 90b, etc. The connection between the conductor 94b and the reference electrodes 63a and 63b is similar.

When measuring the electrical resistance of a cell 9, the cell 9 is supported on the upper surface of the permeable layer 30 in the measurement chamber 100. A tube for supplying a liquid such as culture medium is connected to the supply port 73 of the top member 70, and a tube for discharging the liquid from the measurement chamber 100 is connected to the discharge port 75 of the top member 70. When liquid is injected into the supply flow path 51, the measurement chamber 100 and the measurement flow path 40 are filled with liquid. Furthermore, the liquid is discharged from the discharge flow path 53, thereby appropriately discharging the liquid from the measurement chamber 100. This allows for the exchange (or circulation) of liquid between the first chamber 101 and the second chamber 102 of the measurement chamber 100.

In Figure 3, resistance Rm corresponds to the electrical resistance of the portion of the permeation layer 30 within the measurement chamber 100 and the cells 9 supported in that portion (hereinafter referred to as the "cell portion"). Resistance Rw1 corresponds to the electrical resistance of the liquid between the working electrode 61a and the cell portion (i.e., the first chamber 101 of the measurement chamber 100). Resistance Rw2 corresponds to the electrical resistance of the liquid between the working electrode 61b and the cell portion (i.e., the measurement flow path 40 and the second chamber 102).

In addition, in Figure 3, resistance Rr1 corresponds to the electrical resistance of the liquid between the reference electrode 63a and the cell portion (i.e., the first chamber 101 of the measurement chamber 100). Resistance Rr2 corresponds to the electrical resistance of the liquid between the reference electrode 63b and the cell portion (i.e., the measurement flow path 40 and the second chamber 102).

The power supply 91 applies a voltage between the working electrodes 61a and 61b, and simultaneously, the voltmeter 92 measures the voltage between the reference electrodes 63a and 63b. The exact voltage between the working electrodes 61a and 61b is then calculated from the measured voltage value using a computer (not shown) or by hand, and the electrical resistance between the working electrodes 61a and 61b is calculated from that voltage value. Furthermore, the resistance Rm of the cell portion is calculated from the calculated electrical resistance between the working electrodes 61a and 61b using a computer (not shown) or by hand.

When the power supply 91 applies a voltage between the working electrodes 61a and 61b, oxidation and reduction reactions of the liquid may occur on the surfaces of the working electrodes 61a and 61b, forming an electric double layer. In this case, there is a risk that the output voltage from the power supply 91 will differ from the voltage applied between the working electrodes 61a and 61b. In the case of the culture device 1, reference electrodes 63a and 63b are disposed near the working electrodes 61a and 61b, respectively, inside the measurement chamber 100 or the measurement flow path 40. Therefore, by measuring the voltage between the reference electrodes 63a and 63b and using the measured voltage as the voltage between the working electrodes 61a and 61b, the resistance Rm of the cell portion can be measured accurately.

<Effects>
The culture device 1 is provided with a measurement flow path 40 connected to the measurement chamber 100, and a working electrode 61b is disposed in the measurement flow path 40. Therefore, by applying a voltage between the working electrode 61a disposed in the measurement chamber 100 and the working electrode 61b disposed in the measurement flow path 40, electricity can be passed between the measurement chamber 100 and the measurement flow path 40.

In addition, because the working electrodes 61a, 61b and the reference electrodes 63a, 63b are arranged on the same electrode substrate 20, the cost of forming the electrodes can be reduced compared to when the electrodes are arranged separately on different members. Furthermore, because the working electrodes 61a, 61b are formed on the underside 21 of the electrode substrate 20, variations in the manufacturing process of the working electrodes 61a, 61b and the reference electrodes 63a, 63b (for example, variations in the resistivity of the electrodes, variations in the thickness or dielectric constant of the insulating protective film, etc.) can be reduced.

Furthermore, according to the culture device 1 of this embodiment, the electrode substrate 20 is attached to the upper side of the measurement container 10. This allows the lower surface 21 of the electrode substrate 20 to block the upper opening of the measurement chamber 100 and the upper opening of the measurement flow path 40, while allowing the working electrode 61a and reference electrode 63a to face the measurement chamber 100, and the working electrode 61b and reference electrode 63b to face the measurement flow path 40, respectively.

In addition, because one end of the discharge flow path 53 is connected midway through the measurement flow path 40, the measurement flow path 40 and the discharge flow path 53 can be partially shared. This allows the measurement container 10 to be more compact than if the measurement flow path 40 and the discharge flow path 53 were provided independently.

2. Second embodiment
Next, a second embodiment will be described. In the following description, elements having the same functions as elements already described will be given the same reference numerals or reference numerals with an additional alphabetical character, and detailed description thereof may be omitted.

Figure 4 is a cross-sectional view of a culture device 1a according to the second embodiment. The culture device 1a has a conductive pad 83 (conductive portion) that is electrically connected to the working electrode 61b. The conductive pad 83 is exposed on the outside of the culture device 1a.

The conductive pad 83 is arranged on the upper surface of the first member 11 of the measurement container 10. The conductive pad 83 is formed, for example, by vapor deposition of a conductive metal. The width direction size of the electrode substrate 20 in this embodiment is smaller than that of the measurement container 10. Therefore, the first member 11 of the measurement container 10 has a portion that extends outward in the width direction relative to the electrode substrate 20. The conductive pad 83 is arranged on this extending portion. Furthermore, the conductive pad 83 is electrically connected to the working electrode 61b arranged on the electrode substrate 20 by making contact with the working electrode 61b from above and below.

In this way, by providing the conductive pad 83, the working electrode 61b can be electrically connected to an external device. Furthermore, because the conductive pad 83 is exposed to the outside of the culture device 1a, electrical conductivity of the working electrode 61b can be easily achieved. Although not shown, the conductive pads corresponding to the working electrode 61a and reference electrodes 63a and 63b may be disposed on the upper surface of the first member 11.

Although this invention has been described in detail, the above description is illustrative in all respects and does not limit the invention. It is understood that countless variations not illustrated can be envisioned without departing from the scope of this 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.

REFERENCE SIGNS LIST 1, 1a Culture device 10 Measurement container 11 First member 12 Second member 20 Electrode substrate 21 Lower surface (first surface)
30 Permeation layer 40 Measurement flow path 51 Supply flow path 53 Discharge flow path 61 Measurement flow path 61a, 61b Working electrodes 63a, 63b Reference electrodes 81a, 81b Conduction holes 83 Conduction pads (conductive portions)
100 Measurement room 101 1st room 102 2nd room

Claims (6)

1. A culture device comprising:
A measuring container;
an electrode base material disposed on one side of the measurement container in a first direction;
Equipped with
The measurement container comprises:
a measurement chamber capable of containing a liquid;
a measurement flow path having one end connected to the measurement chamber and extending from the measurement chamber toward one side in the first direction;
and
The electrode substrate is
a first surface facing the other side in the first direction;
a first electrode disposed on the first surface and facing the inside of the measurement chamber;
a second electrode disposed on the first surface and facing into the measurement flow channel;
and
the measurement container includes a permeable layer that allows a liquid to pass through and that separates the measurement chamber into a first chamber on one side in the first direction and a second chamber on the other side in the first direction;
and
The measurement container comprises:
a first member that defines the first chamber;
a second member that defines the second chamber;
and
the transmission layer is disposed between the first member and the second member in the first direction,
The culture device comprises:
a conductive portion electrically connected to the first electrode or the second electrode;
and
A culture device, wherein the conductive portion is arranged outside the electrode base material in a second direction intersecting the first direction. 10. The culture device of claim 1,
The first surface of the electrode base material closes an opening on one side in the first direction of the measurement chamber and an opening on one side in the first direction of the measurement flow path. The culture device according to claim 1 or claim 2,
The measurement container comprises:
a supply flow path connected to the measurement chamber and supplying a liquid to the measurement chamber;
a discharge flow path connected to the measurement chamber and configured to discharge a liquid from the measurement chamber;
A culture device comprising: The culture device of claim 3,
A culture device, wherein one end of the discharge flow path is connected to the middle of the measurement flow path. The culture device according to any one of claims 1 to 4,
The measuring container has a conductive hole into which a probe pin can be inserted,
A culture device, wherein at least one of the first electrode or the second electrode faces the conductive hole. A method for producing the culture device according to any one of claims 1 to 5, comprising:
(a) forming the first electrode and the second electrode on the first surface of the electrode base material;
(b) attaching the electrode base material on which the first electrode and the second electrode are formed in the step (a) to one side in the first direction of the measurement container;
A method for manufacturing a culture device, comprising: