JP7217521B2 - microfluidic device - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication date: January 11, 2019 (Publication date: January 11, 2019) Publications Cell Assay Research Group 2019 Symposium "Current and Future of Cell Assay Technology" Lecture Proceedings

Application of Article 30, Paragraph 2 of the Patent Law Date January 30, 2019 Meeting name, Venue Cell Assay Study Group 2019 Symposium "Current and Future of Cell Assay Technology" National Institute of Advanced Industrial Science and Technology Tsukuba Center Public Auditorium (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture)

Application of Article 30, Paragraph 2 of the Patent Act Website publication date March 1, 2019 to March 14, 2019 Website address https://gakkai-web. net/iee/program/2019/

Application of Article 30, Paragraph 2 of the Patent Law Distribution date: March 1, 2019 and March 15, 2019 (Issue date: March 15, 2019) Publication: The 2019 National Conference of the Institute of Electrical Engineers of Japan, DVD containing a collection of lecture papers - ROM

Article 30, Paragraph 2 of the Patent Law applies Date March 13, 2019 Meeting name and venue 2019 National Conference of the Institute of Electrical Engineers of Japan Hokkaido University of Science (7-15-4-1 Maeda, Teine-ku, Sapporo, Hokkaido)

The present invention relates to microfluidic devices for use in measuring transepithelial electrical resistance (TEER).

Trans-epithelial electric resistance (TEER) is a non-invasive, chronologically measurable index of the barrier function of cell monolayers, and is one of the important evaluation items in drug discovery screening.

Until now, cell culture well plates have been mainly used for TEER measurement, but in recent years, methods using microfluidic devices have also been reported. According to the method using this microfluidic device, expression of membrane tissue specificity is promoted by culturing cells in a microspace within the microfluidic device. can be measured.

For example, Patent Document 1 below discloses a well-type cell culture vessel used for non-invasive quality evaluation of cells. Patent Literature 2 listed below discloses a microfluidic device used to mimic and test the function of body tissue. Further, for example, Non-Patent Documents 1 to 3 below disclose microfluidic devices used for TEER measurement.

WO2012/147463 Japanese Patent Publication No. 2017-513483

M. Odijk et al., “Measuring direct current trans-epithelial electrical resistance in organ-on-a-chip microsystems”, Lab Chip, 2015, 15, 745-752. J. Yeste et al., "Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures", J. Phys. D, 2016, 49(37), 375401. Olivier Y. F. Henry et al., "Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function", Lab Chip, 2017, 17, 2264-2271.

As mentioned in Non-Patent Document 1, even with a microfluidic device, there is a problem that the measurement values differ even if the same cell type is used, depending on the arrangement of the electrodes and cell culture chambers that make up the device. It's becoming Microfluidic devices are susceptible to current density non-uniformity within the microspace, and TEER measurement values tend to vary depending on the device configuration.

An object of the present invention is to provide a microfluidic device for improving the accuracy of TEER measurement.

The present inventors have made intensive studies to achieve the above object, and found that by widening the area in which the cell culture chamber is covered with electrodes in a microfluidic device, the sensitivity fluctuation of TEER measurement is greatly reduced. I found that it can be suppressed. In addition, the inventors have found that by arranging the working electrode and the reference electrode closely together, rapid fluctuations in the sensitivity of the TEER measurement can be suppressed.

That is, the present invention for achieving the above object includes, for example, the following aspects.
(Section 1)
a cell culture chamber;
a first electrode and a second electrode positioned above the cell culture chamber;
a third electrode and a fourth electrode positioned on the underside of the cell culture chamber;
with
a voltage can be applied between the first electrode and the third electrode and between the second electrode and the fourth electrode;
In a plan view, the area where the first electrode or the second electrode overlaps the cell culture chamber is larger than the area where the first electrode or the second electrode does not overlap the cell culture chamber. fluidic device.
(Section 2)
a first transparent substrate on which the first electrode and the second electrode are arranged;
a second transparent substrate provided opposite to the first transparent substrate and having the third electrode and the fourth electrode disposed on the surface thereof;
further comprising
the cell culture chamber is provided between the first transparent substrate and the second transparent substrate;
a permeable membrane provided between the first transparent substrate and the second transparent substrate, on which cells are placed;
a first flow channel provided between the first transparent substrate and the permeable membrane, through which a medium for culturing the cells is fed;
a second flow path provided between the second transparent substrate and the permeable membrane, through which the culture medium is fed;
with
In plan view, the area where the first electrode or the second electrode overlaps the first flow path is larger than the area where the first electrode or the second electrode does not overlap the first flow path. Item 1. The microfluidic device according to item 1, wherein the microfluidic device is wide.
(Section 3)
In a plan view, the area where the third electrode or the fourth electrode overlaps the second flow path is larger than the area where the third electrode or the fourth electrode does not overlap the second flow path. Item 3. The microfluidic device according to item 2, wherein the microfluidic device is also wide.
(Section 4)
A plurality of sets in which the first electrodes and the second electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the first transparent substrate,
The area where the first flow path is covered by the first electrode is wider than the area where the first flow path is covered by the second electrode, and
A plurality of sets in which the third electrodes and the fourth electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the second transparent substrate,
Item 4. The microfluidic device according to item 2 or 3, wherein the area where the second channel is covered by the third electrode is wider than the area where the second channel is covered by the fourth electrode.
(Section 5)
The plurality of first electrodes are arranged substantially symmetrically on the surface of the first transparent substrate at intervals, and the second electrode is arranged between the plurality of first electrodes. ,
The plurality of third electrodes are arranged substantially symmetrically at intervals on the surface of the second transparent substrate, and the fourth electrode is arranged between the plurality of third electrodes. 3. The microfluidic device according to claim 2 or 3.
(Section 6)
Item 4. The microfluidic device according to Item 2 or 3, wherein a plurality of sets of the first electrode and the second electrode adjacent to each other are arranged on the surface of the first transparent substrate.
(Section 7)
Item 7. The microfluidic device according to item 6, wherein a plurality of sets of the third electrode and the fourth electrode adjacent to each other are arranged on the surface of the second transparent substrate.
(Section 8)
Item 8. The microfluidic device according to item 7, wherein a region in which the third electrode and the fourth electrode are not arranged is provided substantially in the center of the second transparent substrate.
(Section 9)
A plurality of sets in which the third electrodes and the fourth electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the second transparent substrate,
Item 7. The microfluidic device according to item 6, wherein the area of the second channel covered by the third electrode is wider than the area of the second channel covered by the fourth electrode.
(Section 10)
10. The microfluidic device of any of Clauses 4, 8 and 9, wherein fluorescence emitted from the fluorescently labeled cells is emitted through the region where the third electrode and the fourth electrode are not arranged.
(Item 11)
Item 1, wherein the ratio of the area where the first electrode or the second electrode overlaps with the cell culture chamber in plan view is in the range of about 65% to about 95% with respect to the area of the cell culture chamber. 4. The microfluidic device according to any one of 3 to 3.

According to the present invention, it is possible to provide a microfluidic device for improving the accuracy of TEER measurement.

1 shows a microfluidic device according to an embodiment of the invention; FIG. (A) is a perspective view and (B) is a top view. 2 is an exploded perspective view of the microfluidic device shown in FIG. 1; FIG. FIG. 2 is a cross-sectional view along line XX of the spatial region R of the microfluidic device shown in FIG. 1; FIG. 2 is a perspective view showing an example of an analysis model applied to a spatial region R of the microfluidic device shown in FIG. 1; FIG. 10 is a diagram showing an electrode shape of pattern AA; (A) is a top view, and (B) is a bottom view. FIG. 10 is a diagram showing the electrode shape of pattern BB; (A) is a top view, and (B) is a bottom view. FIG. 10 is a diagram showing an electrode shape of pattern CC; (A) is a top view, and (B) is a bottom view. FIG. 10 is a diagram showing an electrode shape of pattern DD; (A) is a top view, and (B) is a bottom view. FIG. 10 is a diagram showing the electrode shape of pattern CB; (A) is a top view, and (B) is a bottom view. FIG. 4 is a schematic diagram showing a circuit diagram of a TEER measurement system during sensitivity analysis; 4 is a graph showing the results of sensitivity analysis performed on various electrode shapes. 4 is a graph showing the results of sensitivity analysis for various electrode shapes. 4 is a graph showing the results of sensitivity analysis for various electrode shapes. FIG. 10 is a diagram showing an electrode shape further improved from pattern CC; (A) is a top view, and (B) is a bottom view.

Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. In the following description and drawings, the same reference numerals denote the same or similar components, and redundant description of the same or similar components will be omitted.
[Device configuration]
<Microfluidic device>

FIG. 1 is a diagram showing a microfluidic device according to one embodiment of the present invention. (A) is a perspective view and (B) is a top view. 2 is an exploded perspective view of the microfluidic device shown in FIG. 1. FIG. FIG. 3 is a cross-sectional view of the spatial region R of the microfluidic device shown in FIG. 1 along line XX. An analysis model applied to the spatial region R shown in FIG. 1 will be described later with reference to FIGS. 4 to 9. FIG.

A microfluidic device 10 according to one embodiment of the present invention comprises a cell culture chamber 6 , an upper working electrode 11 , a lower working electrode 12 , an upper reference electrode 13 and a lower reference electrode 14 . The upper working electrode 11 and the upper reference electrode 13 are arranged on the upper side of the cell culture chamber 6 and the lower working electrode 12 and the lower reference electrode 14 are arranged on the lower side of the cell culture chamber 6 .

In order to realize TEER measurement by the four-terminal measurement method described later, a voltage is applied between the upper working electrode 11 and the lower working electrode 12 and between the upper reference electrode 13 and the lower reference electrode 14 during the TEER measurement. be done. The working electrodes 11 and 12 are electrodes for carrying current (for applying voltage), and the reference electrodes 13 and 14 are electrodes for measuring voltage.

As will be understood by those skilled in the art, when a drawing shows a plurality of electrodes, the same voltage is applied to electrodes of the same type described with the same reference numerals. Also, as will be appreciated by those skilled in the art, the four planar electrodes (top working electrode 11, bottom working electrode 12, top reference electrode 13 and bottom reference electrode 14) are arranged so as not to electrically contact each other. are arranged at predetermined intervals.

In this embodiment, the microfluidic device 10 further comprises an upper transparent substrate 1 and a lower transparent substrate 2 . The lower transparent substrate 2 is provided facing the upper transparent substrate 1, and the upper transparent substrate 1, the cell culture chamber 6, and the lower transparent substrate 2 are laminated. An upper working electrode 11 and an upper reference electrode 13 are arranged on the surface of the upper transparent substrate 1 . A lower working electrode 12 and a lower reference electrode 14 are arranged on the surface of the lower transparent substrate 2 .

The cell culture chamber 6 is provided between the upper transparent substrate 1 and the lower transparent substrate 2, and has a permeable membrane 5, a first channel 15 provided in the culture layer 3, and a cell culture chamber 6 provided in the measurement layer 4. and a second flow path 16 . In the following description, the first channel 15 is also called the culture layer channel 15 and the second channel 16 is also called the measurement layer channel 16 .

Culture layer 3 is provided between upper transparent substrate 1 and permeable membrane 5 . A measurement layer 4 is provided between the lower transparent substrate 2 and the transmissive film 5 . The permeable membrane 5 is provided between the culture layer 3 and the measurement layer 4, and cells 90 are placed on its surface. The permeable membrane 5 separates the first flow path 15 and the second flow path 16 so that the culture medium sent to the first flow path 15 and the second flow path 16 can permeate.

The upper transparent substrate 1 , the culture layer 3 and the permeable membrane 5 are provided with a through hole 21 communicating with the first channel 15 and a through hole 22 communicating with the second channel 16 . A medium for culturing cells 90 is fed to the first channel 15 through the through-holes 21 . A culture medium is sent to the second channel 16 through the through holes 22 .

A through hole 23 is provided in the upper transparent substrate 1 . Through holes 24 are provided in the upper transparent substrate 1 , the culture layer 3 and the measurement layer 4 . During TEER measurement, electrode terminals (not shown) for TEER measurement are inserted into the through holes 23 and 24 . The upper working electrode 11 and the upper reference electrode 13 are electrically connected to electrode terminals for TEER measurement through cables passed through the through-holes 23 . The lower working electrode 12 and the lower reference electrode 14 are electrically connected to electrode terminals for TEER measurement through cables passed through the through-holes 24 . The manner in which these electrodes 11 to 14 are electrically connected to electrode terminals through through holes 23 and 24 provided in upper transparent substrate 1 is an example. The electrode terminals may be inserted from the bottom of the microfluidic device 10 through through holes provided in the lower transparent substrate 2 or may be inserted from the side of the microfluidic device 10 . The through-holes 23 and 24 may have any configuration, and these electrodes 11 to 14 may be electrically connected to electrode terminals during TEER measurement.

In the microfluidic device 10 according to the present embodiment, in order to improve the accuracy of TEER measurement, the area where the upper working electrode 11 or the upper reference electrode 13 overlaps the cell culture chamber 6 in plan view (cells The area where the upper working electrode 11 or the upper reference electrode 13 does not overlap the cell culture chamber 6 (the area where the cell culture chamber 6 is not covered by the electrode 11 or the electrode 13) is designed to be wide. Preferably, the ratio of the area where the upper working electrode 11 or the upper reference electrode 13 overlaps the cell culture chamber 6 to the area of the cell culture chamber 6 in plan view is within the range of about 65% to about 95%. Moreover, preferably, the area where the upper working electrode 11 overlaps the cell culture chamber 6 is larger than the area where the upper reference electrode 13 overlaps the cell culture chamber 6 in plan view.

Specifically, in plan view, the area where the upper working electrode 11 or the upper reference electrode 13 overlaps with the first channel 15 (the area where the first channel 15 is covered with the electrode 11 or the electrode 13) is The upper working electrode 11 or the upper reference electrode 13 is configured to be wider than the area that does not overlap the first channel 15 (the area where the first channel 15 is not covered by the electrode 11 or the electrode 13). . Preferably, in plan view, the ratio of the area of the upper working electrode 11 or the upper reference electrode 13 overlapping the first channel 15 to the area of the first channel 15 is within the range of about 65% to about 95%. is. Moreover, preferably, the area of the upper working electrode 11 overlapping the first channel 15 is larger than the area of the upper reference electrode 13 overlapping the first channel 15 in plan view.

Also, preferably, in plan view, the area where the lower working electrode 12 or the lower reference electrode 14 overlaps with the second channel 16 (the area where the second channel 16 is covered with the electrode 12 or the electrode 14) is The area where the lower working electrode 12 or the lower reference electrode 14 does not overlap the second flow path 16 (the area where the second flow path 16 is not covered by the electrode 12 or the electrode 14) is configured to be larger than that. . Preferably, in plan view, the ratio of the area of the lower working electrode 12 or the lower reference electrode 14 overlapping the second channel 16 to the area of the second channel 16 is in the range of about 65% to about 95%. is. In addition, preferably, the area where the lower working electrode 12 overlaps with the second channel 16 is larger than the area where the lower reference electrode 14 overlaps with the second channel 16 in plan view.

The microfluidic device 10 according to this embodiment can be manufactured, for example, by a technique for manufacturing MEMS (Micro Electro Mechanical Systems). The microfluidic device 10 can be manufactured by other microfabrication techniques besides the MEMS technique. The upper transparent substrate 1 and the lower transparent substrate 2 are made of a transparent material such as thermoplastic or glass, and are made of polycarbonate in this embodiment. The four electrodes, upper working electrode 11, lower working electrode 12, upper reference electrode 13 and lower reference electrode 14, are made of a conductive metal such as gold, silver, copper or aluminum, and in this embodiment are made of gold. It is In this embodiment, these four electrodes are formed in thin films. The culture layer 3 and the measurement layer 4 are made of a transparent material, and are made of polydimethylsiloxane (PDMS) in this embodiment. The permeable membrane (or substance-permeable membrane) 5 is a membrane or layer through which the cells 90 do not permeate and substances can diffuse (for example, metabolites and drugs can permeate). Illustratively, the permeable membrane 5 can be made of a transparent material having a porous structure. In another embodiment, a transparent material composed of a large number of linear through holes can be used for the transmissive film 5 . In this embodiment shown as an example of the embodiment of the present invention, the permeable membrane 5 is a porous membrane and is made of transparent polyethylene terephthalate.

Exemplary thicknesses of the upper transparent substrate 1 and the lower transparent substrate 2 are both about 0.1 mm to about 2 mm, preferably both about 1 mm. Exemplary thicknesses of culture layer 3 and measurement layer 4 are both about 10 μm to about 1000 μm, preferably both about 150 μm. An exemplary thickness of the permeable membrane 5 is from about 1 μm to about 100 μm, preferably about 12 μm.
<Overview of TEER measurement>

In TEER measurement, the target cell tissue is first cultured on a permeable membrane. After that, a voltage is applied between electrodes provided above and below the cell monolayer, impedance is measured, and TEER is calculated based on an equivalent circuit. In this embodiment, the TEER measurement uses a four-terminal measurement method that can reduce the influence of measurement errors by incorporating two types of electrodes, one set each of a working electrode and a reference electrode. The four-terminal measurement method is a measurement method that can eliminate the influence of contact impedance and the like and enables highly accurate impedance measurement.

The electrical resistance obtained by the TEER measurement system is the total resistance R _total including the internal fluid resistance, the electrode-fluid interface resistance, the resistance of the permeable membrane used for cell culture, etc. Therefore, R _total itself is regarded as the electrical resistance by the cell. It is not possible. In order to solve this problem, before introducing cells, electrical resistance measurement was performed in the same device under the same conditions such as the internal fluid and electrical resistance measurement system, and the total resistance obtained was R _blank . do. Then, the electrical resistance R _cells caused by cells is represented by the following equation (1).

Also, the electrical resistance is inversely proportional to the cell culture area A _cult . In order to compare measurement results between microfluidic devices and transwells with different cell culture areas, the TEER value is defined by the following equation (2) by normalization.

Therefore, for TEER measurement, it is sufficient to know the total resistance and the cell culture area before and after cell introduction.

In the above description, the case of performing TEER measurement at a single frequency is assumed as an example, and the electrical resistance is measured for the device before introducing cells, but the method of TEER measurement is exemplified above. not limited to Various commercially available TEER measuring devices can be used as the TEER measuring device. Moreover, the microfluidic device according to the present invention can be used not only in commercially available TEER measurement devices but also in impedance measurement devices.
[Apparatus evaluation method and evaluation results]

First, the current density in the microfluidic device is analyzed by the finite element method. Next, the sensitivity analysis of the microfluidic device is performed by comparing the distribution of the sensitivity in the device depending on the electrode shape, and the electrode shape of the microfluidic device suitable for TEER measurement is evaluated.
<Analysis model>

FIG. 4 is a perspective view showing an example of an analysis model applied to the spatial region R of the microfluidic device shown in FIG.

The analysis of the current density targets the spatial region R indicated by the dashed line in the microfluidic device 10 shown in FIG. As illustrated in FIG. 4, the analysis model 50 (51-56) includes four types of planar electrodes (upper working electrode 11, lower working electrode 12, upper reference electrode 13 and lower reference electrode 14) and a cell culture chamber 6 (permeable membrane 5 , culture layer channel 15 and measurement layer channel 16 ) and cell monolayer 90 . The culture layer channel 15 and the measurement layer channel 16 are filled with a medium for culturing the cells 90 . The analysis model 50 (51 to 56) has a major axis dimension L of about 4000 μm and a minor axis dimension W of about 1000 μm.

In this embodiment, five analytical models 50 (51 to 55) having different shapes of planar electrodes are prepared. The current density analysis is performed for each of the five analysis models 51-55 having various electrode shapes shown in FIGS. 5-9 using analysis software such as COMSOL Multiphysics.

5 to 9, the pattern α-β refers to the electrode shape on the upper surface of the analysis model (arranged on the upper side of the cell culture chamber 6). It means that the shape of the electrode on the lower surface (the shape of the electrode arranged on the lower side of the cell culture chamber 6) is the pattern β.

FIG. 5 is a diagram showing the electrode shape of pattern AA. (A) is a top view, and (B) is a bottom view.

The analytical model 51 has an electrode shape of pattern AA. Pattern A is a conventional arrangement in which the areas on both ends of the cell culture chamber 6 are covered with electrodes. The area of the cell culture chamber 6 covered with the electrodes is narrow, and the area not covered with the electrodes is wider than the area covered with the electrodes.

On the upper surface of the analytical model 51 shown in FIG. 5A, the upper working electrode 11 and the upper reference electrode 13 are arranged on the surface of the upper transparent substrate 1 on the left side of the drawing with a space therebetween. It covers the left side of 15. The bottom surface is the same as the top surface. On the lower surface of the analytical model 51 shown in FIG. 5B, the lower working electrode 12 and the lower reference electrode 14 are arranged on the surface of the lower transparent substrate 2 on the right side of the drawing with a space therebetween. It covers the right side of 16.

An exemplary lateral dimension of upper working electrode 11, lower working electrode 12, upper reference electrode 13, and lower reference electrode 14 is about 200 μm. An exemplary lateral dimension of the region of the culture layer channel 15 or measurement layer channel 16 located between these electrodes is about 100 μm. In the exemplified embodiment, the ratio of the area of the upper working electrode 11 or the upper reference electrode 13 overlapping the culture layer channel 15 to the area of the culture layer channel 15 on the upper surface of the analysis model 51 is about 10%. 51, the ratio of the area of the lower working electrode 12 or the lower reference electrode 14 overlapping the measurement layer channel 16 to the area of the measurement layer channel 16 is about 10%.

FIG. 6 is a diagram showing the electrode shape of pattern BB. (A) is a top view, and (B) is a bottom view.

The analytical model 52 has an electrode shape of pattern BB. Pattern B is an arrangement in which a region in which no electrodes are provided (hereinafter also referred to as a cell observation region) is provided in order to enable cell observation by fluorescence, and most of the cell culture chamber 6 is covered with electrodes. The area of the cell culture chamber 6 covered with electrodes is wider than the area not covered with electrodes. Exemplarily, in the analysis model 52, cell observation areas 15c and 16c of approximately 1×1 mm ² are provided approximately in the center of the cell culture chamber 6, and an upper reference electrode 13 and a lower reference electrode having an electrode width of approximately 200 μm. 14 are provided.

On the upper surface of the analytical model 52 shown in FIG. 6(A), on the surface of the upper transparent substrate 1, one side area and the other side area bounded by the area 15c where the upper working electrode 11 and the upper reference electrode 13 are not arranged. In each, an upper working electrode 11 and an upper reference electrode 13 are arranged. The arrangement of the upper working electrode 11 and the upper reference electrode 13 on one side of the upper transparent substrate 1 and the arrangement of the upper working electrode 11 and the upper reference electrode 13 on the other side are substantially symmetrical. .

That is, on the upper surface of the analytical model 52, a plurality of pairs of alternately arranged upper working electrodes 11 and upper reference electrodes 13 are arranged substantially symmetrically (in the illustrated embodiment, substantially symmetrically). The region where the culture layer channel 15 is covered with the upper working electrode 11 is configured to be wider than the region where the culture layer channel 15 is covered with the upper reference electrode 13 .

Exemplary lateral dimensions of upper working electrode 11 and upper reference electrode 13 are about 1200 μm and about 200 μm, respectively. An exemplary lateral dimension of the region 15c located between the upper reference electrodes 13, 13 of the culture layer channel 15 is about 1000 μm. An exemplary lateral dimension of the region of the culture channel 15 located between the upper working electrode 11 and the upper reference electrode 13 is about 100 μm. In the exemplified embodiment, the ratio of the area of the upper working electrode 11 or the upper reference electrode 13 overlapping the culture layer channel 15 to the area of the culture layer channel 15 on the upper surface of the analytical model 52 is about 70%.

The bottom surface is the same as the top surface. On the lower surface of the analytical model 52 shown in FIG. 6(B), on the surface of the lower transparent substrate 2, one side area and the other side area bounded by the area 16c where the lower working electrode 12 and the lower reference electrode 14 are not arranged. In each, a lower working electrode 12 and a lower reference electrode 14 are arranged. The arrangement of the lower working electrode 12 and the lower reference electrode 14 on one side of the lower transparent substrate 2 and the arrangement of the lower working electrode 12 and the lower reference electrode 14 on the other side are substantially symmetrical. .

That is, on the lower surface of the analytical model 52, a plurality of sets of alternately arranged lower working electrodes 12 and lower reference electrodes 14 are arranged substantially symmetrically (in the illustrated embodiment, substantially symmetrically). The area over which the measurement layer channel 16 is covered by the lower working electrode 12 is configured to be larger than the area over which the measurement layer channel 16 is covered by the lower reference electrode 14 .

Exemplary lateral dimensions of lower working electrode 12 and lower reference electrode 14 are approximately 1200 μm and approximately 200 μm, respectively. An exemplary lateral dimension of region 16c located between lower reference electrodes 14, 14 is approximately 1000 μm. An exemplary lateral dimension of the region of measurement layer channel 16 located between lower working electrode 12 and lower reference electrode 14 is about 100 μm. In the illustrated embodiment, the ratio of the area of the lower working electrode 12 or the lower reference electrode 14 overlapping the measurement layer channel 16 to the area of the measurement layer channel 16 on the bottom surface of the analytical model 52 is about 70%.

FIG. 7 is a diagram showing the electrode shape of pattern CC. (A) is a top view, and (B) is a bottom view.

The analysis model 53 has an electrode shape of pattern CC. Pattern C is an electrode arrangement that divides the electrodes while maintaining electrical equivalence. The area where the cell culture chamber 6 is covered with electrodes is wider than the area not covered with electrodes, and the working electrodes 11, 12 and the reference electrode 13, 14 are arranged alternately. No cell observation area is provided. The working electrodes 11, 12 and the reference electrodes 13, 14 are preferably arranged alternately in order, and the width of the working electrodes 11, 12 and the width of the reference electrodes 13, 14 are preferably approximately equal.

Illustratively, in analytical model 53, exemplary lateral dimensions of upper working electrode 11, upper reference electrode 13, lower working electrode 12, and lower reference electrode 14 are approximately 200 μm. An exemplary lateral dimension of the region of the culture layer channel 15 or measurement layer channel 16 located between these electrodes is about 100 μm. In the exemplified embodiment, the ratio of the area of the upper working electrode 11 or the upper reference electrode 13 overlapping the culture layer channel 15 to the area of the culture layer channel 15 on the upper surface of the analysis model 53 is about 65%. On the lower surface of 53, the ratio of the area where the lower working electrode 12 or the lower reference electrode 14 overlaps the measurement layer channel 16 to the area of the measurement layer channel 16 is about 65%.

In the upper surface of the analytical model 53 shown in FIG. 7A, a plurality of pairs of adjacent upper working electrodes 11 and upper reference electrodes 13 are arranged on the surface of the upper transparent substrate 1 . The bottom surface is the same as the top surface. On the lower surface of the analytical model 53 shown in FIG. 7B, a plurality of pairs of adjacent lower working electrodes 12 and lower reference electrodes 14 are arranged on the surface of the lower transparent substrate 2 .

FIG. 8 is a diagram showing the electrode shape of pattern DD. (A) is a top view, and (B) is a bottom view.

The analysis model 54 has an electrode shape of pattern DD. Pattern D is an arrangement that maximizes the area where the cell culture chamber 6 is covered with electrodes. Most of the cell culture chamber 6 is covered with electrodes, and the area of the cell culture chamber 6 covered with electrodes is wider than the area not covered with electrodes. No cell observation area is provided.

On the upper surface of the analytical model 54 shown in FIG. 8(A), on the surface of the upper transparent substrate 1, an upper action An electrode 11 is arranged. The shape of the upper working electrode 11 arranged on one side of the upper transparent substrate 1 and the shape of the upper working electrode 11 arranged on the other side of the upper transparent substrate 1 are substantially symmetrical.

That is, on the upper surface of the analytical model 54 , the plurality of upper working electrodes 11 are arranged substantially symmetrically (in the illustrated embodiment, substantially left-right symmetrical) at intervals on the surface of the upper transparent substrate 1 , and the upper reference electrode 13 are arranged between the plurality of upper working electrodes 11 .

Illustratively, an exemplary lateral dimension of the upper reference electrode 13 is about 100 μm. An exemplary lateral dimension of the region of the culture channel 15 located between the upper working electrode 11 and the upper reference electrode 13 is about 100 μm. In the exemplified embodiment, the ratio of the area of the upper working electrode 11 or the upper reference electrode 13 overlapping the culture layer channel 15 to the area of the culture layer channel 15 on the upper surface of the analytical model 54 is about 95%.

The bottom surface is the same as the top surface. On the lower surface of the analysis model 54 shown in FIG. 8B, on the surface of the measurement layer channel 16, the lower A working electrode 12 is arranged. The shape of the lower working electrode 12 arranged on one side of the lower transparent substrate 2 and the shape of the lower working electrode 12 arranged on the other side of the lower transparent substrate 2 are substantially symmetrical.

That is, on the lower surface of the analytical model 54 , the plurality of lower working electrodes 12 are arranged substantially symmetrically (in the illustrated embodiment, substantially symmetrical) on the surface of the lower transparent substrate 2 , and the lower reference electrode 14 are arranged between the plurality of lower working electrodes 12 .

Illustratively, an exemplary lateral dimension of the lower reference electrode 14 is about 100 μm. An exemplary lateral dimension of the region of measurement layer channel 16 located between lower working electrode 12 and lower reference electrode 14 is about 100 μm. In the illustrated embodiment, the ratio of the area where the lower working electrode 12 or the lower reference electrode 14 overlaps the measurement layer channel 16 to the area of the measurement layer channel 16 on the bottom surface of the analytical model 54 is about 95%.

FIG. 9 is a diagram showing the electrode shape of pattern CB. (A) is a top view, and (B) is a bottom view.

The analytical model 55 has an electrode shape of pattern CB. In the pattern CB, a cell observation area is provided approximately in the center of one side of the cell culture chamber 6 (lower side in the illustrated embodiment). The area of the cell culture chamber 6 covered with electrodes is wider than the area not covered with electrodes. In addition, on one side of the cell culture chamber 6 where the cell observation area is provided, the area where the cell culture chamber 6 is covered with the electrodes is configured to be wider than the cell observation area.

In the upper surface of the analytical model 55 shown in FIG. 9A, a plurality of pairs of adjacent upper working electrodes 11 and upper reference electrodes 13 are arranged on the surface of the upper transparent substrate 1 .

On the lower surface of the analytical model 55 shown in FIG. 9(B), on the surface of the lower transparent substrate 2, one side area and the other side area bounded by the area 16c where the lower working electrode 12 and the lower reference electrode 14 are not arranged. In each, a lower working electrode 12 and a lower reference electrode 14 are arranged. The arrangement of the lower working electrode 12 and the lower reference electrode 14 on one side of the lower transparent substrate 2 and the arrangement of the lower working electrode 12 and the lower reference electrode 14 on the other side are substantially symmetrical. .

That is, on the lower surface of the analytical model 55, a plurality of sets of alternately arranged lower working electrodes 12 and lower reference electrodes 14 are arranged approximately symmetrically (in the illustrated embodiment, approximately symmetrically). The area over which the measurement layer channel 16 is covered by the lower working electrode 12 is configured to be larger than the area over which the measurement layer channel 16 is covered by the lower reference electrode 14 .
<Summary of sensitivity analysis and evaluation criteria>

In electrical resistance measurement, not all regions of the object to be measured equally contribute to the measured resistance value, and the regions between the electrodes and in the vicinity of the electrodes have a greater effect on the measured value. This is because the current density distribution in the object to be measured differs depending on the shape and arrangement of the electrodes. Therefore, it is required to appropriately design them and control the current flowing through the object to be measured.

In current density analysis using the finite element method, the sensitivity s is defined as a measure of the contribution of each minute element to the measured value. Sensitivity is calculated for each minute element from the current density in that minute element and is represented by the following equation (3).

Here, vector J1 is the current density in each microelement when the current _I is applied between the working electrodes, and vector J2 is the current density when the current _I is applied between the reference electrodes. FIG. 10 is a schematic diagram showing a circuit diagram of the TEER measurement system during sensitivity analysis. ( _A ) shows the time of J1 measurement, and (B) shows the _time of J2 measurement. In this embodiment, the object 99 to be measured in the drawing means the cell culture chamber 6 in which the cells 90 and medium are set. Specifically, the measurement object 99 includes the culture layer 3 provided with the culture layer channel 15 containing the medium, the permeable membrane 5 having the cells 90 placed on the surface, and the measurement layer channel 16 containing the medium. It means a configuration including a provided measurement layer 4 . The electrical resistance R to be measured is expressed by the following equation (4), where V is the volume and ρ is the electrical resistivity of the minute element dv.

These equations show that the greater the absolute value of sensitivity, the greater the influence of that minute element on the measured value.

TEER is evaluated under the assumption that a uniform current density distribution is formed in a defect-free monolayer of cells. In practice, uniform cell culture is not necessarily achieved, and this corresponds to the change in electrical resistivity in the above equation (4). Therefore, if the cell culture conditions in the region corresponding to a certain microelement are different from those in the surrounding region, the electrical resistivity of this microelement will change, but the effect on the measurement value will be only the sensitivity of the microelement. Increased or decreased. For example, if the cells are densely packed and have a high electrical resistivity in a highly sensitive microelement, the measured electrical resistance will be very large. In other words, the measurement uncertainty due to the cell state and the systematic error due to the device structure combine to fluctuate the measured value, resulting in the TEER measurement error. Therefore, by making the sensitivity constant over the entire area of the cell culture chamber, measurement errors due to the device structure can be avoided, and highly accurate TEER measurement can be achieved. This can be expressed by the following equation (5).

That is, the ideal shape is a shape in which the sensitivity is constant over the entire region. At this time, each minute element affects the measured value equally. Also, extreme values of sensitivity in certain micro-elements of the cell culture chamber should be avoided as they lead to large measurement errors as mentioned above. Therefore, it is desirable that the difference between the maximum value and the minimum value of sensitivity is small. Therefore, the sensitivity fluctuation s ^* is defined as the criterion for evaluating the superiority or inferiority of the shape by the following equation (6).

where s _max , s _min and _save are the maximum, minimum and average sensitivity, respectively. The large non-uniformity of the current density, which depends on the electrode shape, causes a problem in the measurement accuracy of TEER.
<Analysis results and discussion>

FIG. 11 is a graph showing the results of sensitivity analysis performed on various electrode shapes. 12 and 13 are graphs showing the results of sensitivity analysis for various electrode shapes.

The analysis results are shown in FIGS. 11 to 13 as sensitivity distributions along the axial centerline of the cell culture chamber 6 . Referring to the analysis results, the sensitivity is stable in the region where the cell culture chamber 6 is covered with the electrodes (working electrodes 11, 12 or reference electrodes 13, 14), but the working electrodes 11, 12 and the reference electrodes 13, 13 are stable. It is confirmed that there is a sharp change in sensitivity at the boundary between 14 .

Looking at the analysis results for each electrode shape, the pattern CC in which the working electrodes 11 and 12 and the reference electrodes 13 and 14 are closely arranged has an excellent sensitivity variation of 0.97%. In addition, the sensitivity variation in pattern BB and pattern DD is approximately 10% or less, and it is confirmed that the sensitivity is greatly improved compared to the sensitivity variation due to pattern AA, which is the conventional arrangement. be. Pattern BB has a cell observation area, and is useful in that fluorescent staining of cells can be easily observed.

In this way, it is confirmed that the sensitivity fluctuation is suppressed in all electrode shapes of pattern BB, pattern CC, and pattern DD, and the sensitivity is improved compared to the conventional pattern AA. be done.

On the other hand, pattern CC has the least variation in sensitivity and is excellent, but since the electrode covers the area corresponding to the cell observation area, fluorescence staining observation of cells is not easy. This is because, in many cases, an inverted microscope is used for staining observation of cells. Therefore, according to the pattern CB in which the cell observation area is provided on the lower surface of the microfluidic device of the pattern CC, the sensitivity fluctuation can be greatly suppressed, and the fluorescence staining observation of the cells is also possible, which is useful. . This pattern CB has a sensitivity variation of 4.51%, achieving a sensitivity variation of less than 5%. In addition, by comparing the sensitivity variation of the pattern CC and the sensitivity variation of the pattern CB, it was found that if either the upper surface side or the lower surface side of the cell culture chamber 6 has the electrode shape of the pattern C, the sensitivity is significantly increased. confirmed to improve.

Based on the above considerations, sensitivity fluctuations can be greatly suppressed by widening the area where the cell culture chamber 6 is covered with the electrodes. Also, by closely arranging the working electrodes 11 and 12 and the reference electrodes 13 and 14, rapid fluctuations in sensitivity can be suppressed.

As a guideline for designing the electrode shape of the microfluidic device 10, the upper working electrode 11 or the upper reference electrode 13 covers the cell culture chamber 6, and the upper working electrode 11 or the upper reference electrode 13 covers the cell culture chamber 6. It has been shown to be configured to be wider than the uncovered area. The bottom surface is the same as the top surface.
[Other forms]

Although the present invention has been described in terms of specific embodiments, the present invention is not limited to the embodiments described above.

In the above-described embodiment, as an example, the application of the microfluidic device 10 is measurement of transepithelial electrical resistance (TEER), but the application of the microfluidic device 10 is not limited to this. The microfluidic device 10 can be used not only for measuring transepithelial electrical resistance (TEER) but also for measuring electrical resistance of various cell layers such as epithelial cells and vascular endothelial cells.

A cell observation area may be provided in the analysis model 53 having the electrode shape of pattern CC described above.

FIG. 14 is a diagram showing an electrode shape further improved from pattern CC. (A) is a top view, and (B) is a bottom view. The analysis model 56 shown in FIG. 14 is a model in which a cell observation area is provided substantially in the center of one side (lower surface side in the illustrated embodiment) of the analysis model 53 having the electrode shape of pattern CC. The area of the cell culture chamber 6 covered with electrodes is wider than the area not covered with electrodes. In addition, on one side (lower side) of the cell culture chamber 6 where the cell observation area 16c is provided, the area where the cell culture chamber 6 is covered with the electrodes is configured to be wider than the cell observation area 16c. there is The cell observation area may be provided on one side (lower side) of the cell culture chamber 6, may be provided on the other side (upper side), or may be provided on both sides (upper and lower sides). Illustratively, in the analysis model 56, a cell observation area 16c of about 1×1 mm ² is provided in the lower side of the cell culture chamber 6 and substantially in the center.

In the electrode shape of pattern C described above, as shown in FIG. (The order of electrodes 11 and 13 is the order when viewed from the left side of FIG. 7), but the order of upper working electrode 11 and upper reference electrode 13 is different in adjacent pairs. may For example, the electrodes 11 and 13 are arranged in the order of the electrodes 11 and 13 in the first group, the electrodes 11 and 13 are arranged in the order of the electrodes 13 and 11 in the second group, and the electrodes 11 and 13 are arranged in the order of the electrodes 13 and 11 in the first group. 2 may be arranged side by side. The same is true for lower working electrode 12 and lower reference electrode 14 .

The shape of the four types of planar electrodes (upper working electrode 11, lower working electrode 12, upper reference electrode 13, and lower reference electrode 14) in the area covering the cell culture chamber 6 is not limited to the illustrated strip shape. For example, with respect to the electrode shape of pattern C described above, the shape of the planar electrode is not limited to the alternate arrangement of the strip-shaped upper working electrode 11 and the upper reference electrode 13 shown in the figure, but is circular and arranged substantially concentrically. , elliptical or rectangular upper working electrodes 11 and upper reference electrodes 13 may be alternately arranged. Moreover, the shape of the electrodes is not limited to a plane, and may be a three-dimensional shape as long as it covers most of the cell culture chamber 6 .

A specific method of current density analysis by the finite element method is described below. COMSOL Multiphysics version 5.4, AC/DC module was used as software for current density analysis. The analysis procedure is as follows from procedure 1 to procedure 4 below.

Step 1: Set the shape of the analysis model in COMSOL.

The elements of the analysis model (electrodes x many, medium x 2, permeable membrane, cell layer) were all combinations of rectangular parallelepiped elements. The dimensions and physical properties of each element are as follows.
・Electrode 200×1000×0.29[μm], σ=61.6E6[S/m], ε=1 (dimensions, electrical conductivity, dielectric constant)
・Medium 4000×100×150[μm], σ=1.5[S/m], ε=78
・Permeable membrane 4000×1000×12[μm], R _m =30[Ωcm ² ], ε=3
・Cell layer 4000×1000×10[μm], TEER=250[Ωcm ² ], ε=10

Step 2: Set boundary conditions.

・Working electrode (+ side)
Terminal (set so that the total of multiple working electrodes on the + side becomes 1A)
For example, if there are 5 working electrodes on the top surface, set each as a 0.2A terminal.
・Working electrode (- side)
Ground (0V)
・Reference electrode Floating potential

In addition, the electrode surface which is electrically equipotential was made to be equipotential by giving a restraining condition. For example, if there are five working electrodes on the top surface, set their potentials as Vn as V1=V2=V3=V4=V5.

Step 3: Divide the analysis model into meshes and perform analysis.

Step 4: Calculate the sensitivity variation from the minimum, maximum and mean values of the sensitivity in the z-direction central section of the cell layer.

1 upper transparent substrate (first transparent substrate)
2 lower transparent substrate (second transparent substrate)
3 culture layer 4 measurement layer 5 permeable membrane 6 cell culture chamber 10 microfluidic device 11 upper working electrode (first electrode)
12 lower working electrode (third electrode)
13 upper reference electrode (second electrode)
14 lower reference electrode (fourth electrode)
15 culture layer channel (first channel)
16 measurement layer channel (second channel)
21-24 through-hole 50 (51-55) analysis model 90 cell (cell monolayer)

Claims (11)

a cell culture chamber;
a first electrode and a second electrode positioned above the cell culture chamber;
a third electrode and a fourth electrode positioned on the underside of the cell culture chamber;
with
a voltage can be applied between the first electrode and the third electrode and between the second electrode and the fourth electrode;
In a plan view, the area where the first electrode or the second electrode overlaps the cell culture chamber is larger than the area where the first electrode or the second electrode does not overlap the cell culture chamber. fluidic device. a first transparent substrate on which the first electrode and the second electrode are arranged;
a second transparent substrate provided opposite to the first transparent substrate and having the third electrode and the fourth electrode disposed on the surface thereof;
further comprising
the cell culture chamber is provided between the first transparent substrate and the second transparent substrate;
a permeable membrane provided between the first transparent substrate and the second transparent substrate, on which cells are placed;
a first flow channel provided between the first transparent substrate and the permeable membrane, through which a medium for culturing the cells is fed;
a second flow path provided between the second transparent substrate and the permeable membrane, through which the culture medium is fed;
with
In plan view, the area where the first electrode or the second electrode overlaps the first flow path is larger than the area where the first electrode or the second electrode does not overlap the first flow path. 2. The microfluidic device of claim 1, wherein the is also wide. In a plan view, the area where the third electrode or the fourth electrode overlaps the second flow path is larger than the area where the third electrode or the fourth electrode does not overlap the second flow path. 3. The microfluidic device of claim 2, wherein the is also wide. A plurality of sets in which the first electrodes and the second electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the first transparent substrate,
The area where the first flow path is covered by the first electrode is wider than the area where the first flow path is covered by the second electrode, and
A plurality of sets in which the third electrodes and the fourth electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the second transparent substrate,
4. The microfluidic device according to claim 2, wherein the area covered by the third electrode of the second channel is wider than the area covered by the fourth electrode of the second channel. The plurality of first electrodes are arranged substantially symmetrically on the surface of the first transparent substrate at intervals, and the second electrode is arranged between the plurality of first electrodes. ,
The plurality of third electrodes are arranged substantially symmetrically at intervals on the surface of the second transparent substrate, and the fourth electrode is arranged between the plurality of third electrodes. , a microfluidic device according to claim 2 or 3. 4. The microfluidic device according to claim 2, wherein a plurality of sets of said first electrode and said second electrode adjacent to each other are arranged on the surface of said first transparent substrate. 7. The microfluidic device according to claim 6, wherein a plurality of sets of said third electrode and said fourth electrode adjacent to each other are arranged on the surface of said second transparent substrate. 8. The microfluidic device according to claim 7, wherein a region in which said third electrode and said fourth electrode are not arranged is provided substantially in the center of said second transparent substrate. A plurality of sets in which the third electrodes and the fourth electrodes are alternately arranged are arranged substantially symmetrically at intervals on the surface of the second transparent substrate,
7. The microfluidic device according to claim 6, wherein the area of said second channel covered by said third electrode is larger than the area of said second channel covered by said fourth electrode. 10. The microfluidic device of any of claims 4, 8 and 9, wherein fluorescence emitted from the fluorescently labeled cells is emitted through the region where the third and fourth electrodes are not arranged. . 3. The ratio of the area of the first electrode or the second electrode that overlaps the cell culture chamber to the area of the cell culture chamber in plan view is in the range of about 65% to about 95%. 4. The microfluidic device according to any one of 1 to 3.

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