JP2008263858A - Apparatus and chip for assaying cell response - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for assaying cell response to a medicament, and to provide a chip for the apparatus. <P>SOLUTION: The apparatus for assaying cell response comprises microelectrodes, capable of assaying the electrical condition change of only single cell placed on a chip; a cell-nonadhesive wall encircling the microelectrodes so as to locate the cell on the microelectrodes; a layer filled with a cell culture solution; a mechanism for refluxing the cell culture solution and adding, as necessary, a medicament thereto, annular reference electrodes located above the microelectrodes; a mechanism for measuring and recording cell potential which uses both the microelectrodes and the reference electrodes; and a mechanism for optically measuring the conditions of the single cell placed on the chip. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an apparatus for measuring a cell response to a drug, electrical stimulation, and the like, and a cell response measuring chip.

  Bioassays are often used to observe changes in the state of cells and the response of cells to drugs and the like. Conventional bioassays often use cultured cells in general. In this system, since the assay is performed using a plurality of cells, the average value of the value of the cell population has been observed as if it were a characteristic of one cell.

  In reality, however, cells rarely have a synchronized cell cycle in the population, and each cell expresses a protein in a different cycle. For this reason, the problem of fluctuations always accompanies when analyzing the results of responses to stimuli.

  That is, since there is a response fluctuation universally possessed by the cell reaction mechanism itself, only an average response can always be obtained. In order to solve these problems, techniques such as synchronized culture have been developed, but using a group of cells that are always in the same stage means that such cells must always be supplied. It is an obstacle to the widespread spread of bioassays.

  In addition, there are two types of stimulation (signal) for cells: those given by the amount of signal substances, nutrients and dissolved gas contained in the solution around the cells, and those by physical contact with other cells and cell-cell interactions. Therefore, it was actually difficult to judge fluctuations.

  The problem of physical contact of cells and cell-cell interaction can be solved to some extent by performing a bioassay on a cell mass such as a tissue fragment. However, in this case, unlike cultured cells, a uniform cell mass cannot always be obtained. Therefore, there are problems that the obtained data varies and information is buried in the group.

In order to measure an information processing model in which each cell of a cell group is a minimum structural unit, the inventors of the present application describe a cell in a specific space as disclosed in JP-A-2006-94703 (Patent Document 1). Configure multiple cell culture compartments to be confined in the arrangement, connect adjacent compartments with grooves or tunnels that do not allow cells to pass through, and optionally groove or tunnel or cell culture We proposed an aggregate cell microarray (bioassay chip) with a structure having multiple electrode patterns for measuring cell potential changes in the compartment.
JP 2006-94703 A

  In conventional bioassays, cells were either treated as tissue fragments or treated as one cell like cultured cells. If the number of cells is too large, the information obtained is averaged as described in the section of the prior art, and there is a problem that a response to a drug or the like cannot be obtained accurately. When cells are used one by one, cells that originally function as cells of a multicellular tissue are used as separated and independent cells, so that the influence of cell-to-cell interaction does not appear. Again, there are problems in obtaining accurate drug response or bioassay data. Therefore, the cell potential and cell morphology can be accurately measured with one cell, and if necessary, the cell potential and cell morphology of one cell can be accurately measured by placing cells in the vicinity and being affected by the interaction between cells. It is important to develop devices and systems that can be used.

  In order to solve the above-mentioned problems, the present invention keeps one cell confined in a specific spatial arrangement, and forms a microelectrode for measuring the cell potential of the cell in the spatial arrangement area. An electrode is disposed at a position away from the surface of the microelectrode in the Z-axis direction within a range including the space arrangement region, and the cell culture medium is refluxed to the cells so that the cell activity can be maintained. In order to measure the cell potential more accurately, the distance in the XY axis direction and the distance in the Z axis direction between the microelectrode and the comparison electrode can be adjusted. In addition, a means for adding a drug acting on the cells is added.

  Furthermore, in order to be able to evaluate the influence of interaction between cells, it is possible to appropriately arrange cells around one cell confined in a specific spatial arrangement, or to make one cell have a specific spatial arrangement. A configuration in which the configurations confined inside are regularly arranged. In addition, it is possible to evaluate the reaction by applying different drugs to each cell in the same environment with a constant temperature layer in a configuration where one cell is confined in a specific spatial arrangement in a constant temperature layer. The configuration is as follows.

  The cell potential of one cell confined in a specific spatial arrangement and the response of the cell to the drug can be accurately measured, and evaluation including the influence of interaction between cells can be performed.

  FIG. 1 is a perspective view schematically showing an example of the structure of a cell response measuring apparatus according to an embodiment of the present invention. FIG. 2 is a schematic diagram showing a cross section of the cell response measuring apparatus shown in FIG. 1 viewed on the stage of the optical observation apparatus.

Reference numeral 100 denotes a cell response measuring apparatus, which is mainly composed of components constructed on the transparent substrate 1. The transparent substrate 1 is an optically transparent material such as a glass substrate or a silicon substrate. Reference numeral 2 denotes a microelectrode, which is, for example, a transparent electrode made of ITO and disposed on the transparent substrate 1. Reference numeral 2 ′ denotes a lead wire for the microelectrode 2. Reference numerals 3 ₁ , 3 ₂ , 3 _3, and 3 ₄ denote agarose gel walls, which are arranged around the microelectrode 2 via spaces 4 ₁ , 4 ₂ , 4 _3, and 4 ₄ . The walls 3 ₁ , 3 ₂ , 3 _3, and 3 ₄ of the agarose gel are notched at the center to form a space that serves as a cell storage portion. A microelectrode 2 is disposed on a transparent substrate 1 in a space serving as a cell storage portion formed by walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ formed of agarose gel, and one cell 100 ₀ can be stored on this. . 5 is a comparative electrode by gold or platinum rings, holding space upper comprising a cell housing unit, or upper cell 10 _0, in a distance of X-Y axis direction of the distance and the Z-axis direction to an arbitrary position adjustable Is done. 5 ₁ the other end is fixed to one end of reference electrode 5 at the lead portion for holding the reference electrode 5 is held in the position adjusting device 6 of the comparative electrode 5. Lead portion 5 ₁ also serves as a lead wire of the reference electrode. One end of the adjusting device 6 is fixed to the substrate 1. 6 ₁ is a drive device in the Z-axis direction, is provided in the position adjusting device 6. Drive units ₆₁ in the Z-axis direction to move the reference electrode 5 in the vertical direction via the lead portion 5 ₁ for holding the reference electrode 5 is driven by a drive signal Dz given by the computer 9 to be described later. 6 ₂ is a drive in the Y-axis direction, is provided in the position adjusting device 6. Y-axis direction of the drive device 6 ₂ moves the reference electrode 5 in the Y-axis direction via the lead portion 5 ₁ for holding the reference electrode 5 is driven by a drive signal Dy given by the computer 9 to be described later. 6 ₃ is a driving device for the X-axis direction, is provided in the position adjusting device 6. Drive device 6 ₃ in the X-axis direction to move the reference electrode 5 in the X-axis direction via the lead portion 5 ₁ for holding the reference electrode 5 is driven by a drive signal Dx given by the computer 9 to be described later. Thickness and diameter of the reference electrode 5 can be mounted on the position adjusting device 6 to change over the lead portions 5 _1, if necessary. Reference numeral 7 denotes a wall surrounding the periphery, which surrounds a space serving as a cell storage portion formed by the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ made of agarose gel. Reference numerals 8 ₁ and 8 ₂ denote pipes for refluxing the cell culture solution into the region inside the wall 7. In the example shown in the figure, the culture solution is supplied from the pipe 8 ₁ extended to the bottom of the substrate 1. , cultures are discharged from the pipe 8 a _second position close to the upper surface of the wall 7. A pipe 8 ₃ is connected near the outlet of the culture solution of the pipe 8 ₁ for supplying the culture solution, and a drug desired to act on the cells is supplied through this pipe 8 ₃ .

9 is a personal computer, a cellular potential measuring recorded between a lead portion 5 ₁ for holding the reference electrode 5 and the lead wires 2 'of the microelectrode 2. An operation signal Ms from the operator is applied to the personal computer 9. Operator, measurement of the response of cell cellular potential, to the agent, prior to recording, the cells placed in culture in the area inside the wall 7 before storing the cells 10 ₀ on the microelectrode 2, noise The voltage is measured, and the optimum values of the thickness and diameter of the reference electrode 5 are searched. In addition, the operator operates the drive signals Dx, Dy, and Dz while the culture solution is simply put in the region inside the wall 7, and the distance in the XY axis direction and the Z axis direction of the comparison electrode 5. To find the optimum position.

A microelectrode 2 is arranged on a substrate 1 in a space serving as a cell storage portion formed by cutting out the central portions of the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ by agarose gel. cell 10 ₀ is placed. The mounting surface of the cell 10 ₀ microelectrode 2 better to cells such collagen greased materials that help to adhere to the electrode surface. On the other hand, since agarose gel is non-adhesive for cells, even if the height of the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ is the same as that of the cells, the cells 100 ₀ move over the walls. There is no. In addition, the spaces 4 ₁ , 4 ₂ , 4 ₃ and 4 ₄ around the cell storage part formed by cutting out the central parts of the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ by the agarose gel are the cell size. , be made smaller than is the cell 10 ₀ does not move slip through the space portion 4 _1, 4 _2, 4 ₃ and 4 _4. Therefore, the cell 10 ₀ is stably held on the microelectrode 2 while being exposed to the cell culture medium refluxed to the region inside the wall 7 by the pipes 8 ₁ and 8 ₂ .

An example of the main size of the structure of the cell response measuring apparatus 100 shown in FIG. 1 is as follows. This is an example in which the cell size is 10 μmφ. The transparent substrate 1 has a size of 100 mm × 150 mm, the microelectrode 2 has a size of 8 μm × 8 μm, and the individual sizes of the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ by agarose gel are 20 μm × 20 μm × 10 μm (high The space 4 ₁ , 4 ₂ , 4 ₃ and 4 _{4 has} a width of 2 μm, and the space serving as a cell storage part formed by the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ made of agarose gel is a cylindrical shape of 12 μmφ. The outer shape of the wall 7 is 5 mm × 5 mm, the height is 5 mm, the reference electrode 5 is a platinum wire with a thickness of 50 μmφ, and the diameter of the ring is 1 mmφ. Here, the microelectrode 2 is a square of 8 μm × 8 μm, but the entire walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ and the spaces 4 ₁ , 4 ₂ , 4 ₃ and 4 ₄ are formed by agarose gel. It is good also as a 10-micrometer (phi) circular electrode used as the storage part of the cell comprised by width.

Figure 2 is a schematic view showing a cross section viewed cell response measurement apparatus 100 is put on the stage of optical observation apparatus at the center of the cell 10 ₀ shown in FIG. 1, when observing the behavior of the cell by the optical system An overview is shown. Reference numeral 21 denotes an XY stage of the optical observation device, which is optically transparent and is moved to an arbitrary position by the XY drive device 20 in accordance with a signal given by the personal computer 9. In Figure 2, shows a state where the supply of the culture liquid 11 of the cell 10 ₀ is supplied from the pipe 8 ₁ inside the region of the wall 7, are discharged from the pipe 8 a _second position close to the upper surface of the wall 7.

Reference numeral 22 denotes a light source for a phase contrast microscope or differential interference microscope, and a halogen-based lamp is generally used. Reference numeral 23 denotes a band-pass filter that transmits only light having a specific wavelength from light from a light source for observation of a stereoscopic microscope such as a phase difference. For example when the observation of the cells 10 _0, it is possible to prevent damage of the cells 10 ₀ by the use of light in a narrow band near the wavelength 700 nm. Reference numeral 24 denotes a shutter, which has a function of blocking light irradiation during image measurement, such as when the XY stage 21 is moved. A condenser lens 25 introduces a phase difference ring for phase difference observation, and introduces a polarizer for differential interference observation. A cell response measuring device 100 formed on the substrate 1 is placed on the XY stage 21, and the XY stage 21 is moved by an XY driving device 20, thereby moving an arbitrary position of the cell response measuring device 100. Observe and measure. Presence state of the cell 10 ₀ in the cell response measurement apparatus 100 is observed by the objective lens 26. The focal position of the objective lens 26 can be moved by the driving device 27 in accordance with a signal from the personal computer 9. The objective lens 26 having a magnification of 40 times or more can be used.

Also, for the fluorescent observation, the light is transmitted through only the excitation light wavelength by the band-pass filter 32 from the light source 31, only the excitation light when viewed by the shutter 33 is controlled so as to be irradiated to the cell 10 _0. Excitation light that has passed through the shutter 33 is irradiated to the cells 10 ₀ by the dichroic mirror 34. At this time being observed by the objective lens 26, a phase difference image or differential interference image of the cell 10 ₀ by transmitted light from the light source 22, the cell 10 ₀ by the excitation light from the light source 31 is a fluorescent image emitted . Only the phase contrast microscope image or the differential interference microscope image is observed by the camera 37 by the dichroic mirror 35 and the band pass filter 36 that reflect light having the same wavelength as that transmitted through the band pass filter 23. On the other hand, the fluorescent image can be observed with the camera 40 by selectively transmitting only the wavelength band of the fluorescence observation by the mirror 38 and the band pass filter 39 of the light passing through the objective lens 26. The phase difference image or differential interference image taken by the camera 37 is analyzed by the image processing function of the personal computer 9, and the movement amount of the XY stage 21, the height control of the objective lens 26, and the focusing can be performed. . The camera 37 can be a 1/200 high-speed camera and can be used for pulsation image analysis.

FIG. 3A, FIG. 3B, and FIG. 3C are diagrams showing the concept of noise signal evaluation related to cell potential measurement. Respectively, the distance in the X direction on the horizontal axis and the microelectrode 2 and the reference electrode 5, is changed in the Y and Z directions, between the lead 5 ₁ leader line 2 'and the reference electrode 5 of the microelectrode 2 on the vertical axis It is a figure which shows the magnitude | size of a noise signal when the amplitude of the voltage which can be detected is taken. It can be seen that the noise sharply decreases at positions Lx, Ly, and Lz from arbitrary base points. This data is measured prior to the measurement, and drive signals Dx, Dy and Dz are given from the personal computer 9 to adjust the distance in the XY and Z-axis directions of the comparison electrode 5 at the optimum position. Try to measure.

  Hereinafter, a configuration example of the cell response measuring apparatus 100 of the present invention and a specific measurement example using the same will be described.

Figure 4 is a diagram showing one example of how to use when using a cell response measuring apparatus 100 described in FIGS. 1 and 2, to measure the potential at the time of beating cardiomyocyte 10 _0. As can be seen in comparison with FIG. 1, the cell containing the walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ and the widths of the spaces 4 ₁ , 4 ₂ , 4 ₃ and 4 ₄ by agarose gel is stored. the microelectrode 2 parts besides cardiomyocytes 10 ₀ is placed, by agarose gel walls 3 _1, 3 _2, 3 ₃ and 3 whole ₄ and the space portion 4 _1, 4 _2, 4 ₃ and 4 ₄ Both are the same except that the myocardial cells 10 ₁ , 10 ₂ ,..., 10 ₈ are dispersedly arranged in the inner region of the wall 7 in the periphery. In this configuration, it cardiomyocytes 10 _1, 10 _2, ---, the potential at the placed cardiomyocyte 10 ₀ beats the microelectrode 2 according to the situation of the distributed arrangement of 10 ₈ drawer microelectrodes 2 It is measured as the potential between the lead 5 ₁ of the reference electrode 5 and the line 2 '.

On the other hand, FIG. 5 is a diagram showing using a cell response measuring apparatus 100 described in FIGS. 1 and 2, an example of another One use when measuring the potential at the time of beating cardiomyocytes 10 ₀ . As can be seen in comparison with FIG. 4, in the configuration of FIG. 4, the entire walls 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ and the periphery of the spaces 4 ₁ , 4 ₂ , 4 ₃ and 4 ₄ are formed by agarose gel. cardiomyocytes 10 ₁ inside the region of the walls 7, 10 _2, ---, 10 ₈ cardiomyocytes 10 ₁ of these only are distributed is 10 _2, ---, strictly controlled the position of 10 ₈ It wasn't possible. In contrast, in the arrangement of FIG. 5, cardiomyocytes 10 ₁ not only cardiomyocytes 10 _0, 10 _2, ---, walls 3 ₁ by agarose gel all 10 _8, 3 _2, 3 ₃ and 3 ₄ and overall space 4 _1, 4 _2, 4 ₃ and so surrounded by 4 _4, cardiomyocytes 10 _1, 10 _2, ---, 10 positions ₈ may strictly controlled. Furthermore, in the configuration of FIG. 5, cardiomyocyte 10 _0, 10 _1, 10 _2, ---, 10 ₈ each microelectrode 2 ₀ placed is 2 _1, 2 _2, ---, 2 ₈ ( In the drawing, reference numerals are omitted) and the microelectrodes between the cardiomyocytes 10 ₀ , 10 ₁ , 10 ₂ , ---, 10 _{8 and} the common reference electrode 5 provided in common. 2, the cardiac potential of each of the cardiomyocytes 10 ₀ , 10 ₁ , 10 ₂ ,..., 10 _{8 is} the lead-out line 2 ′ ₀ , 2 ′ ₁ , 2 ′ of each microelectrode 2. _2, --- it can be measured as a potential between the 2 _'8 lead 5 ₁ comparison electrode 5.

6 (A) is a state in which cardiomyocytes 10 ₀ are isolated, i.e., FIG. 4, the state shown in FIG. 5 cardiomyocytes 10 ₁ shown in, 10 _2, ---, 10 ₈ the absence (Fig. 1 ) is a diagram showing the potential at the time of beating cardiomyocytes 10 ₀ at. FIG. 6 (B) 4 to the periphery of cardiac myocytes 10 _0, cardiomyocytes 10 _1, 10 ₂ shown in FIG. 5, ---, in the two cells state where only one has been placed among the 10 ₈ it is a diagram illustrating the potential at the time of beating cardiomyocyte 10 _0. FIG 6 (C) is 4 at the periphery of cardiac myocytes 10 _0, cardiomyocytes 10 _1, 10 ₂ shown in FIG. 5, ---, cardiomyocytes 10 ₀ 9 cells with all is distributed in 10 ₈ It is a figure which shows the electric potential at the time of pulsation. FIG 6 (A) As can be seen by comparing ~ a (C), is the most unstable pulsation state orphaned cardiomyocyte 10 _0, a 2 cellular state seen little improvement, 9 cell state stability It can be seen that the pulsating state.

  FIGS. 7A to 7C are diagrams showing experimental results in which the relationship between the heartbeat interval fluctuation (CV%) and the number of cardiomyocytes forming a network is evaluated with respect to the arrangement pattern of cells in which the cardiomyocytes form a network. It is. On the left side of the figure, the horizontal axis represents the number of cardiomyocytes forming the network, the vertical axis represents the fluctuation of the heartbeat interval (CV%), and the right side of the figure shows the arrangement pattern of the cells forming the cell network. This is shown as a wall structure made by the agarose gel 3. 7A shows a case where the cell arrangement pattern is a lattice, FIG. 7B shows a case where the cell arrangement pattern is radial, and FIG. 7C shows a case where the cell arrangement pattern is linear. Is the case. In order to make the cell arrangement pattern radial, in FIG. 7B, the wall formed by the outer agarose gel 3 at the diagonal position is rotated by 45 °, and the agarose gel 3 at the central position at the diagonal position is formed. The wall to be made has a space part in the diagonal direction. It can be seen that fluctuations in the heartbeat interval (CV%) can be improved by increasing the number of cardiomyocytes forming the network, regardless of the cell arrangement pattern.

  FIG. 7D shows characteristics obtained by overlapping the characteristics shown in FIGS. 7A to 7C. It can be seen that the characteristics shown in FIGS. 7A to 7C are substantially overlapped. That is, it can be seen that the heartbeat interval fluctuation (CV%) of the cardiomyocytes forming the network does not depend on the cell arrangement pattern.

FIG. 8 shows the time schedule for evaluating the effect of the drug received by the cardiomyocytes at the top, and the waveform (left side) and pulsation of the pulsation of the cardiomyocytes at the time points (a) to (e) shown in the time schedule. It is a figure which shows distribution (right side) in the lower stage. The waveform of the pulsation is obtained by taking the signal intensity on the vertical axis and taking time on the horizontal axis, and the pulsation distribution is obtained by taking the frequency on the vertical axis and the pulsation cycle on the horizontal axis. 8, the 4 cells in the configuration shown in FIG. 5 arranged in a grid pattern, while while supplying the culture solution from the pipe 8 ₁ is discharged from the pipe 8 ₂ reflux culture in the region of the wall 7, the pipe 8 through a pipe 8 ₃ connected to _1, shows the results of experiments by adding antipsychotic haloperidol (HPL) in culture to assess the effect of the drug.

Time point (a) is a control state, which is a reference state for evaluating the effect of the drug. During the control period, the culture solution is supplied from the pipe 8 _{1 in} FIG. 5 and discharged from the pipe 8 ₂ . After the control state has been continued for 6 minutes, antipsychotic haloperidol (HPL) was added culture solution is to be circulated to the area of the wall 7 through a pipe 8 _3. Time point (b) is 1 minute after HPL administration, and time point (c) is 5 minutes after HPL administration. After continuing the HPL administration 7 minutes, the addition of antipsychotic haloperidol through a pipe 8 ₃ (HPL) stopped, only the culture solution is to be circulated to the area of the wall 7. That is, cells exposed to HPL are washed. Time point (d) is 6 minutes after HPL cleaning, and time point (e) is 12 minutes after HPL cleaning.

  In the control state, the signal waveform of the pulsation at the time point (a) is stable, and the pulsation distribution is within the range of approximately 1 second in the pulsation period. The control state was continued for 6 minutes, and then HPL administration was started. Looking at the time point (b) 1 minute after the administration of HPL, the pulsation signal waveform is disturbed, and there is a time when large deformation is shown as shown by the arrows. Further, HPL administration is continued, and when looking at the time point (c) 6 minutes after HPL administration, the disturbance of the pulsation signal waveform is larger, and the situation of large deformation as shown by the arrows is increasing. After the HPL administration is continued for 7 minutes, the HPL administration is stopped and only the culture solution is refluxed to wash the HPL of the cells. Looking at the time point (d) 6 minutes after the HPL washing, the pulsation period is extended, but the disturbance of the signal waveform is reduced. Further, when the HPL cleaning is continued and the time point (e) 12 minutes after the HPL cleaning is observed, the pulsation period is shortened and the disturbance of the signal waveform is reduced.

  FIGS. 9 (A) and 9 (B) are diagrams for comprehensively explaining changes in pulsation fluctuations of cardiomyocytes during HPL administration / washing process, and FIG. 9 (A) shows the time of control, HPL administration and washing process. The fluctuation of the pulsation cycle according to the progress is shown, and the value in the final state of each process is shown by the downward triangle. FIG. 9B is a diagram showing values in the final state of the control, HPL administration, and washing process when the pulsation cycle is taken on the horizontal axis and fluctuation of the pulsation cycle is taken on the vertical axis.

  As can be seen from FIG. 9A, the effect of HPL increases with the lapse of time of HPL administration, and the pulsation state becomes unstable. That is, the fluctuation of the pulsation cycle increases. Then, even when HPL administration is stopped, only the culture solution is refluxed, and the HPL of the cells is washed, the fluctuation of the pulsation cycle continues for a while, and the pulsation cycle is continued by continuing washing. Fluctuation of the falls. That is, the pulsation state gradually recovers due to the medium exchange. However, as can be seen from FIG. 9 (B), when looking at the final state of each process of control, HPL administration, and HPL washing, the fluctuation of the pulsation cycle and pulsation cycle was affected by HPL administration by HPL washing. Is reduced, but does not return to its original state as indicated by the thick arrows.

FIG. 10 shows a time schedule for evaluating the effect of the same drug as described in FIG. 8 by arranging 9 cells in a lattice shape in the configuration shown in FIG. It is a figure which shows the waveform (left side) and pulsation distribution (right side) of the pulsation of the cardiac muscle cell in a)-(d) at the lower stage. The pulsation waveform is obtained by taking the signal intensity on the vertical axis and taking time on the horizontal axis, and the beat distribution is obtained by taking the frequency on the vertical axis and the pulsation cycle on the horizontal axis. Cells were discharged from the pipe 8 ₂ reflux culture in the region of the wall 7 while supplying the culture solution from the pipe 8 ₁ against, and, through a pipe 8 ₃ connected to the pipe 8 _1, antipsychotics The result of the experiment which adds the haloperidol (HPL) to a culture solution and evaluates the effect of a chemical | medical agent is shown.

  Time points (a) to (d) shown in FIG. 8 and FIG. 10 are (a) the control state, (b) one minute after HPL administration, (c) five minutes after HPL administration, ) 6 minutes after HPL washing after HPL administration is continued for 7 minutes, after washing the cells exposed to HPL.

  When the time points (a) to (c) in FIG. 8 are compared with the time points (a) to (c) in FIG. 10, in FIG. 10, since 9 cells are arranged in a lattice pattern, It is observed that the change in the basic pattern of pulsation due to is small, that is, the change in the period of pulsation is small, but the peak appearing immediately after the original pulsation waveform is large. Comparing FIG. 8 (d) and FIG. 10 (d), in FIG. 10 (d), it is observed that both the pulsation period and the pulsation waveform are close to the control state.

In FIG. 11, following the evaluation of the effect of the drug on the time schedule described with reference to FIG. 10, HPL re-administration in which HPL was administered, and HPL re-irrigation in which HPL was washed following this HPL re-administration were performed. The evaluation of the effect of the drug in the case is shown in the top row, and the pulsation waveform (left side) and pulsation distribution (right side) of the myocardial cells at the time points (e) to (g) shown in the time schedule are shown in the bottom row. . The time point (d) in FIG. 11 is the same time point as the time point (d) in FIG. Also in FIG. 11, the waveform of pulsation is obtained by taking the signal intensity on the ordinate and taking time on the abscissa, and the pulsation distribution taking the frequency on the ordinate and taking the pulsation cycle on the abscissa. It is. Cells were discharged from the pipe 8 ₂ reflux culture in the region of the wall 7 while supplying the culture solution from the pipe 8 ₁ against, and, through a pipe 8 ₃ connected to the pipe 8 _1, antipsychotics The result of the experiment which adds the haloperidol (HPL) to a culture solution and evaluates the effect of a chemical | medical agent is shown.

  Time points (e) to (g) shown in FIG. 11 are (e) 1 minute after re-administration of HPL, (f) 5 minutes after re-administration of HPL, and (g) is re-administration of HPL. Is continued for 7 minutes, and then the HPL rewash was 6 minutes after washing the cells exposed to HPL again.

  When the time points (e) and (f) in FIG. 11 are compared with the time points (b) and (c) in FIG. 10, the effect of the drug by re-administration of HPL on the cells is the drug by the first administration of HPL. It can be seen that this is far greater than the effect that has on cells. That is, at time points (e) and (f) in FIG. 11, the signal waveform deformation is much larger than that at the time points (b) and (c) in FIG. ing. On the other hand, the pulsation signal waveform and pulsation cycle 6 minutes after HPL rewashing shown in time (g) in FIG. 11 and the pulsation signal waveform and pulsation cycle in the control state shown in time (a) in FIG. Is similar.

  12 (A) and 12 (B) are diagrams for comprehensively explaining changes in pulsation fluctuation of cardiomyocytes during HPL administration / washing process, HPL re-administration / rewashing process, and FIG. 12 (A) is a control, The fluctuation of the pulsation cycle according to the passage of time of the HPL administration and washing process and the subsequent HPL re-administration / re-washing process is shown, and the values in the final state of each process are shown by downward triangles. In FIG. 12B, the horizontal axis represents the pulsation cycle, and the vertical axis represents fluctuation of the pulsation cycle, the control, the HPL administration and washing process, and the final state of the subsequent HPL re-administration / re-washing process. It is a figure which shows the value in.

  As can be seen from FIG. 12A, the effect of HPL increases with the lapse of time of HPL administration, and the pulsation state becomes unstable. That is, the fluctuation of the pulsation cycle increases. Then, even when HPL administration is stopped, only the culture solution is refluxed, and the HPL of the cells is washed, the fluctuation of the pulsation cycle continues for a while, and the pulsation cycle is continued by continuing washing. Fluctuation of the falls. That is, the pulsation state gradually recovers due to the medium exchange. Then, the effect of HPL appears very strongly by the re-administration of HPL, and the fluctuation of the pulsation cycle greatly increases. Next, stopping HPL re-administration, refluxing only the culture medium, and re-washing the HPL of the cells greatly improves the fluctuation of the pulsation cycle. By continuing the re-washing, fluctuation of the pulsation cycle is controlled. To the same extent. That is, the recovery of the pulsating state by exchanging the medium has a great effect in re-washing.

  As can be seen from FIG. 12 (B), however, when the influence of the drug on the cells is evaluated by a cell group in which 9 cells are arranged in a lattice, the 4 cells shown in FIG. 9 (B) are arranged in a lattice. Compared with the evaluation result by the arranged cell group, the result of washing and re-washing returns to the almost same state as the control state. That is, the pulsation cycle and the fluctuation of the pulsation cycle in the final state of the process of control, HPL washing, and HPL re-washing fall within a narrow range, and the pulsation cycle and pulsation cycle in the final state of the HPL administration and HPL re-treatment processes Fluctuation falls within a narrow range. This is because the final state of each process of the control, HPL administration, and HPL washing shown in FIG. 8 (B) is also affected by HPL washing, but the influence of HPL administration is reduced. It is very different from not returning to the state.

  FIGS. 13A, 13B, and 13C focus on the above-described cardiomyocyte grouping effect and analyze the pulsation rhythm of the cardiomyocytes using the cell response measuring apparatus 100 described in FIG. It is a figure explaining an example of application. That is, focusing on the acquisition of stability even in a small cell population consisting of several cells, it is possible to evaluate QT prolongation syndrome by analyzing the pulsatile waveform by electrical measurement and measuring the ion channel level response to drugs. The level can be understood and applied to drug screening.

FIG. 13A is a diagram showing an example of a heart pulsation waveform (electrocardiogram), and shows a normal electrocardiogram waveform on the left side and an electrocardiogram waveform developing QT prolongation syndrome on the right side. Since QT prolongation syndrome can be induced by lethal arrhythmia induction or antiarrhythmic drugs, it is important to be able to evaluate QT prolongation syndrome in drug screening. FIG. 13B is a diagram showing a measurement example of a pulsation waveform by electrical measurement of cultured cardiomyocytes, and shows a normal waveform by the extracellular potential recording method on the left side and a waveform of QT prolongation syndrome on the right side. As can be seen by comparing FIG. 13 (A) and FIG. 13 (B), in the electrical measurement of cultured cardiomyocytes, (1) translation of the waveform is similar to the electrocardiogram, (2) Na ⁺ , K ⁺ , Ca ^{2 +} Measurement of channel action, (3) Repolarization waveform shows T-wave-like shape, and QT extension can be evaluated. FIG. 13 (C) obtains the pulsatile waveform by electrical measurement of the composition of the cell response measurement apparatus 100 by nine cultured cardiomyocytes shown in FIG. 5 (on the left), the antiarrhythmic drugs were applied via the pipe 8 ₃ It is a figure which shows the QT extension detection result (right side) by the induced | guided | derived drug screening.

  FIGS. 14A and 14B are diagrams for explaining one of points to be noted in the measurement using the above-described cardiomyocyte grouping effect. FIG. 14A shows a configuration in which 8 × 8 (= 64) cells can be arranged inside the wall 7 of the cell response measuring apparatus 100 described in FIG. It is a peak waveform figure of voltage which shows the result of having measured the propagation of the pulsation of the cardiac muscle cell from channel CH1 to CH8 using a piece of cells. FIG. 14B is a diagram showing a result of analyzing the propagation time between each microelectrode in a row in which eight cells are placed on the microelectrodes arranged uniformly. As can be seen from FIGS. 14A and 14B, the propagation time between cells varies greatly. Therefore, in the measurement, it is important to make measurement using one cell in the group together with stabilization using the grouping effect of the cardiomyocytes.

  Furthermore, although description with reference to the drawings is omitted, in evaluating the effect of the drug received by the cell, it is important to add a continuous reflux system that does not stimulate the cell, but in the examples of the present invention, the drug is It will act on the cells after being injected into the reflux system of the culture solution, and the stimulation to the cells can be reduced.

  FIG. 15 is a diagram for explaining an example of an application for optically analyzing the pulsation rhythm of cardiomyocytes of a group of 20 cells using the cell response measuring apparatus 100 described in FIG. In FIG. 15, as shown by assigning numbers 1 to 20 to the right shoulder, 20 cells were arranged and pulsatile image analysis was performed using a 1/30 camera. The average change in luminance of 20 cells was measured, and the first point was displayed as 0.

FIG. 16 is a perspective view showing an embodiment of a cell response measuring device 200 as an opposite electrode to the cell response measuring device 100 described in FIG. That is, the cell response measuring apparatus 100 described with reference to FIG. 5 is arranged in a region surrounded by the wall 7 so that a plurality of cells can maintain a predetermined relationship, and a common culture solution is supplied to all these cells. It was supposed to be. On the other hand, in FIG. 16, the measuring devices 100 ₁ , 100 ₂ , 100 ₃ ,..., 100 ₉ having the cell response measuring device 100 described in FIG. did. Of course, also in FIG. 16, the measurement devices 100 ₁ , 100 ₂ , 100 ₃ ,..., 100 ₉ having the cell response measurement device 100 described in FIG. . In this embodiment, under the same environment given by the common thermostatic layer 200, it is possible to easily evaluate the cell reaction under the conditions set here in each cell response measuring device 100. In the example shown in FIG. 16, the figure is complicated, so the reference numerals of the individual components are omitted.

  The cell response measuring apparatus 100 described in FIG. 1 and FIG. 5 evaluates the behavior of one cell under a common culture solution, the behavior of a cell in a cell population, and the behavior of individual cells in a cell population. The cell response measuring apparatus described in FIG. 16 can be said to be a single cell behavior under a common environment or a cell behavior in a cell population. It can be said that this example is suitable for evaluating the behavior of individual cells within a cell population.

  FIGS. 17A to 17D are diagrams illustrating specific examples of the transparent substrate 1, the microelectrode 2, and the lead-out line 2 ′ of the microelectrode 2 described in FIG. 1. 17A is a micrograph showing a plan view, FIG. 17B is an enlarged micrograph showing the microelectrode portion (left side), an explanatory diagram (right side), and FIG. FIG. 17D is a stereomicroscopic image of the microelectrode portion, and is a cross-sectional view of the micrograph shown in FIG.

  17 (A) to 17 (D), it is possible to perform evaluation for a large number of cardiomyocyte groups at once when a network formed by cardiomyocytes is linear in the cell arrangement pattern shown in FIG. 7 (C). It has been devised. That is, four patterns in which eight microelectrodes 2 are linearly arranged in the vertical direction and four patterns in which eight microelectrodes 2 are linearly arranged in the horizontal direction are configured. In this structure, the connected lead line 2 'is drawn to the periphery of the substrate. In this embodiment, eight groups of cells can be evaluated at a time if different cell groups are associated with each of the eight patterns.

  In order to realize this embodiment, first, an ITO film 41 for forming the microelectrode 2 and the lead-out line 2 ′ is formed on the entire surface of the substrate 1. Thereafter, as shown on the right side of FIG. 17B, the peripheral ITO film is formed as a conductive pattern in which the microelectrode 2 and the lead wire 2 ′ are integrated, and the microelectrode 2 (for example, 4 μmφ) and the lead wire 2 ′ (for example, the width) 4 μm) is removed by the etched portions 42 and 43 by etching the narrow width (for example, 1 μm) of the peripheral portion along the conductive pattern. As a result, the conductive pattern in which all the microelectrodes 2 and the lead-out line 2 'are integrated is cut off from the ITO film 41, and the remaining ITO film 41 remains on the substrate 1 as an electrically connected conductor. Next, an insulating film 47 made of polyimide is formed on the entire surface including the conductive pattern in which the microelectrode 2 and the lead line 2 ′ cut out from the ITO film 41 by etching are integrated. As a result, as shown in FIG. 17C, the peripheral etching portions 42 and 43 along the conductive pattern in which the microelectrode 2 and the lead wire 2 ′ removed by etching are integrated are filled with an insulating film 47 made of polyimide. At the same time, the entire surface is covered with an insulating film 47 made of polyimide. Next, as shown in FIGS. 17C and 17D, the insulating film 47 made of polyimide at a position corresponding to the microelectrode 2 is removed, and a cell placement film made of Pt / Pt-black is formed on the microelectrode 2. 46 is formed. Naturally, the microelectrode 2 and the cell placement film 46 are electrically connected.

  In this embodiment, the ITO film on the entire surface other than the pattern in which the microelectrode 2 and the lead wire 2 'are integrated can be used as a ground layer. Therefore, no signal interference occurs between the adjacent microelectrodes 2 and the lead-out line 2 '.

  Looking at the form of industrial use related to the cell response measuring device according to the present invention, from the standpoint of using a cell response measuring device such as a researcher or a pharmaceutical company, and the position of a manufacturer supplying a chip for the cell response measuring device Can be used. From the standpoint of use, it is easy to supply a chip for a cell response measurement device that contains cardiomyocytes in a space that serves as a cell storage portion of the cell response measurement device. However, since the cardiomyocytes stored in the cell storage space cannot survive for a long time even in the state where the culture solution is put in the chip state, the manufacturer supplying the chip has a short period of use. There may be a supply of chips in a limited form or a supply of chips consisting of the elements of the substrate portion shown in FIG. In the case of supplying a chip, it is left to the user to store the cardiomyocytes in the space serving as the cell storage unit of the cell response measuring device and to assemble the whole.

It is the perspective view which showed typically an example of the structure of the cell response measuring apparatus which concerns on the Example of this invention. FIG. 2 is a cross-sectional view of the cell response measuring apparatus shown in FIG. 1 mounted on the stage of the optical observation apparatus and viewed in the depth direction at the longitudinal center position. (A) is a diagram showing an example of noise signal evaluation relating to cell potential measurement, (B) is the distance between the microelectrode and the reference electrode as a parameter, the horizontal axis is the size of the ring diameter of the reference electrode, and the vertical axis is A characteristic diagram when a signal / noise ratio (S / N) of a voltage signal that can be detected between the lead wire of the microelectrode and the lead of the reference electrode is taken. (C) shows the size of the diameter of the ring of the reference electrode. As parameters, the horizontal axis represents the distance between the microelectrode and the reference electrode, and the vertical axis represents the signal / noise ratio (S / N) of the voltage signal that can be detected between the lead wire of the microelectrode and the lead of the comparison electrode. FIG. (A) is a figure which shows typically the measurement result which accommodated one cardiomyocyte in the space used as the cell storage part of the cell holding section, and measured the pulsation of the cardiomyocyte, and (B) shows the cell holding section. Several cardiomyocytes are inserted in the periphery of the space that serves as the cell storage part, and the cardiomyocytes and peripheral cardiomyocytes are in contact with each other's cells by forming protrusions and forming gap junctions. It is a figure which shows typically the measurement result which measured the pulsation of the myocardial cell. Using a cell response measuring apparatus 100 described in FIGS. 1 and 2, it is a diagram showing an example of another One use when measuring the potential at the time of beating cardiomyocyte 10 _0. (A) is a figure which shows the electric potential at the time of the pulsation of the cardiomyocyte 10 _{0 in} the state where the cardiomyocyte 10 ₀ is isolated. 6 (B) is a diagram showing the potential at the time of beating cardiomyocytes 10 ₀ in 2 cell state in which one of the cells at the periphery of cardiac myocytes 10 ₀ is located. FIG 6 (C) is a diagram showing the potential at the time of beating cardiomyocytes 10 ₀ 9 cells state 9 cells in the periphery of cardiac myocytes 10 ₀ is distributed. (A)-(C) is a figure which shows the experimental result which evaluated the relationship with the number of the cardiomyocytes which form a network regarding the arrangement | positioning pattern of the cell which a cardiomyocyte forms a network about fluctuation | variation (CV%) of a heartbeat interval. . The time schedule for evaluating the effect of the drug received by the cardiomyocytes is shown at the top, and the pulsatile waveform (left side) and pulsation distribution (right side) at the time points (a) to (e) shown in the time schedule FIG. (A), (B) is a figure which comprehensively explains the pulsation fluctuation change of the myocardial cells in the HPL administration / washing process. The time schedule for evaluating the effect of the drug similar to that described in FIG. 8 by arranging 9 cells in a lattice shape in the configuration shown in FIG. 5 is shown at the top, and the time points (a) to ( It is a figure which shows the waveform (left side) and pulsation distribution (right side) of the pulsation of the cardiac muscle cell in d) in the lower stage. Following the evaluation of the effect of the drug on the time schedule described with reference to FIG. 10, the HPL re-administration in which HPL is administered, and the HPL re-washing in which HPL is washed following this HPL re-administration It is the figure which shows evaluation of an effect on the uppermost stage, and shows the waveform (left side) and pulsation distribution (right side) of the pulsation of a myocardial cell at the time points (e) to (g) shown in the time schedule. (A), (B) is a figure which explains comprehensively the pulsation fluctuation change of the cardiac muscle cell in a HPL administration / washing process and a HPL re-administration / rewashing process. (A), (B), and (C) are examples of applications to the rhythm analysis of cardiomyocytes using the cell response measuring apparatus 100 described in FIG. FIG. (A) And (B) is a figure explaining one of the points of caution in the measurement using the grouping effect of a cardiomyocyte. It is a figure explaining an example of the application which optically analyzes the pulsation rhythm of the cardiac muscle cell of the cell group which consists of 20 cells using the cell response measuring apparatus 100 demonstrated in FIG. It is a perspective view which shows the Example of the cell response measuring apparatus 200 used as the counter electrode with the cell response measuring apparatus 100 demonstrated in FIG. (A)-(D) is a figure explaining the specific example of the multi-electrode board | substrate including the transparent substrate 1, the microelectrode 2, and the lead wire 2 'of the microelectrode 2 demonstrated in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Substrate, 2 ... Microelectrode, 2 '... Lead wire of microelectrode 2 3 ₁ , 3 ₂ , 3 ₃ and 3 ₄ ... Wall by agarose gel, 4 ₁ , 4 ₂ , 4 ₃ and 4 ₄ ... Space 5 ... Comparison electrode by platinum ring, 5 ₁ ... Lead portion for holding comparison electrode 5, 6 ... Position adjusting device, 7 ... Wall surrounding the periphery, 6 ₁ ... Drive device in Z axis direction, 6 ₂ ... Y axis direction of the drive unit, 6 ₃ ... X-axis direction of the driving device, Dx, Dy and Dz ... drive signals, 8 _1, 8 _2, 8 ₃ ... pipe, 9 ... PC, Ms ... PC operation signal, 10 _0, 10 ₂ , 10 _3- , 10 ₉ ... cardiomyocytes, 20 ... XY drive device, 21 ... stage of optical observation device, 22 ... light source, 23 ... band-pass filter, 24 ... shutter, 25 ... condenser lens, 26 ... Objective lens 27 ... Drive device 31 ... Light source 32, 36, 39 ... Bandpa Filter, 33 ... shutter, 34, 35 ... dichroic mirrors, 37 and 40 ... camera, 41 ... ITO film, 42, 43 ... etching unit, 46 ... cell mounting 置膜, 47 ... Polyimide an insulating film, 200 ... thermostat.

Claims (22)

Transparent substrate,
A microelectrode disposed on the transparent substrate,
A non-cell-adhesive wall covering the periphery of the microelectrode so that one cell is located on the microelectrode;
A wall formed on the transparent substrate for filling a cell culture solution around the cells;
A culture medium reflux means for refluxing the cell culture medium;
Means for adding a drug acting on the cells to the culture medium;
An annular reference electrode located above the microelectrode;
Means for measuring and recording a cell potential using a lead wire connected to the microelectrode and a lead wire connected to the comparison electrode; and optically measuring the state of one cell placed on the transparent substrate means,
A cell response measuring apparatus comprising:   2. The cell response measuring apparatus according to claim 1, wherein means for adding a drug acting on the cells is added to the culture medium reflux means for refluxing the cell culture medium.   The cell response measuring apparatus according to claim 1, further comprising a means for adjusting a positional relationship between the microelectrode and the comparison electrode.   The cell response measuring apparatus according to claim 1, wherein the non-cell-adhesive wall is divided into a plurality of areas around the microelectrode. Transparent substrate,
A plurality of microelectrodes disposed on the transparent substrate;
A non-cell-adhesive wall covering each of the microelectrodes such that one cell is located on each of the plurality of microelectrodes;
A wall formed on the transparent substrate for filling a cell culture solution around the cells on the plurality of microelectrodes;
A culture medium reflux means for refluxing the cell culture medium;
Means for adding a drug acting on the cells to the culture medium;
An annular reference electrode positioned above the plurality of microelectrodes;
Means for measuring and recording a plurality of cell potentials using a plurality of lead lines connected to the plurality of microelectrodes and a lead line connected to the comparison electrode;
A stage that is driven in the X-Y direction and places the transparent substrate; and means for optically measuring the state of cells on the transparent substrate placed on the stage;
A cell response measuring apparatus comprising:   6. The cell response measuring apparatus according to claim 5, wherein means for adding a drug acting on the cells is added to the culture medium reflux means for refluxing the cell culture medium.   The cell response measuring apparatus according to claim 5, further comprising means for adjusting a positional relationship between the plurality of microelectrodes and the comparison electrode.   6. The cell response measuring apparatus according to claim 5, wherein the plurality of non-cell-adhesive walls are divided into a plurality of areas around the plurality of microelectrodes. Microelectrodes placed on a transparent substrate,
A non-cell-adhesive wall covering the periphery of the microelectrode so that one cell is located on the microelectrode;
A wall formed on the transparent substrate for filling a cell culture solution around the cells;
A culture medium reflux means for refluxing the cell culture medium;
Means for adding a drug acting on the cells to the culture medium;
An annular reference electrode located above the microelectrode;
A cell response measuring element comprising:
A common transparent substrate comprising a plurality of the cell response measurement elements,
A thermostat in which the upper and lower walls for storing the plurality of cell response measurement elements are transparent,
Means for measuring and recording each cell potential using a lead wire connected to each microelectrode of the plurality of cell response measuring elements and a lead wire connected to the comparison electrode; and the common transparent substrate Means for optically measuring the state of one cell of the cell response measuring element disposed above;
A cell response measuring apparatus comprising:   The cell response measuring apparatus according to claim 9, wherein means for adding a drug acting on the cells is added to the culture medium reflux means for refluxing the cell culture medium.   The cell response measuring apparatus according to claim 9, further comprising means for adjusting a positional relationship between the microelectrode and the comparison electrode.   The cell response measuring apparatus according to claim 9, wherein the non-cell-adhesive wall is divided into a plurality of areas around the microelectrode. Transparent substrate,
A microelectrode disposed on the transparent substrate,
A non-cell-adhesive wall surrounding the electrode so that one cell is located on the microelectrode;
A wall formed on the transparent substrate for filling a cell culture solution around the cells;
An annular reference electrode located above the microelectrode, and a lead wire connected to the microelectrode and a lead wire connected to the comparison electrode,
A cell response measuring chip comprising:   The cell response measurement chip according to claim 13, further comprising means for adjusting a positional relationship between the microelectrode and the comparison electrode.   The cell response measuring chip according to claim 13, wherein the non-cell-adhesive wall is divided into a plurality around the electrode. Transparent substrate,
A plurality of microelectrodes disposed on the transparent substrate;
A non-cell-adhesive wall covering each of the microelectrodes such that one cell is located on each of the plurality of microelectrodes;
A wall formed on the transparent substrate for filling a cell culture solution around the cells on the plurality of microelectrodes;
An annular reference electrode located above the plurality of microelectrodes, a plurality of lead wires connected to the plurality of micro electrodes, and a lead wire connected to the comparison electrodes,
A cell response measuring chip comprising:   The cell response measurement chip according to claim 16, further comprising means for adjusting a positional relationship between the plurality of microelectrodes and the comparison electrode.   The cell response measuring chip according to claim 16, wherein the non-cell-adhesive wall is divided into a plurality of areas around the microelectrode. Microelectrodes placed on a transparent substrate,
A non-cell-adhesive wall covering the periphery of the microelectrode so that one cell is located on the microelectrode;
A wall formed on the transparent substrate for filling a cell culture solution around the cells;
An annular reference electrode located above the microelectrode, and a lead wire connected to the microelectrode and a lead wire connected to the comparison electrode,
A cell response measurement chip in which a plurality of cell response measurement elements are formed on a common transparent substrate.   The cell response measuring chip according to claim 19, further comprising means for adjusting a positional relationship between the plurality of microelectrodes and the comparison electrode.   The cell response measuring chip according to claim 19, wherein the cell non-adhesive wall is divided into a plurality of areas around the microelectrode. A multi-electrode substrate comprising a plurality of conductive patterns comprising microelectrodes arranged on a transparent substrate and lead lines connected to the microelectrodes,
The conductive pattern, which is the microelectrode and a lead line connected to the microelectrode, is formed by etching the periphery of the conductive pattern with a narrow width from the transparent electrode formed on the entire surface of the transparent substrate. A multi-electrode substrate in which the transparent electrode portion excluding the conductive pattern is used as a common ground layer for the plurality of conductive patterns.

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