JPH0862209A - Cell potential measuring apparatus - Google Patents

Abstract

PURPOSE: To acurately and efficiently measure potential by arranging an integrat ed cell setting device having an electric connection means to apply electrical signals to minute electrodes while fetching the electrical signals from the minute electrodes. CONSTITUTION: A cell as sample is set on a cell setting part of an integrated cell setting device 1 and a plurality of minute electrodes contact the cell. A stimulation signal from a computer 30 is applied to the cell via a D/A converter 32 and an isolator 33 referring to an image of the cell obtained by an optical observation means 20. In other words, the stimulation signal is applied between two points selected from among 64 minute electrodes of the integrated cell setting device 1. An induced potential between the minute electrodes and the potential of a culture liquid is inputted to the computer 30 via 64 channels of highly sensitive amplifiers 34 and A/D converters 31. Then, the potential is outputted to a display device 23 or the like through a necessary signal processing. Measurement of a spontaneous potential without application of the stimulation signal is performed in the same way.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell potential measuring device used in the field of so-called electroneurophysiology for measuring potential changes associated with nerve cell activity.

[0002]

2. Description of the Related Art In recent years, medical examination of nerve cells and possibility of application as an electric element have been actively conducted. An action potential is generated when a nerve cell is activated.
This action potential is caused by a change in the ion concentration inside and outside the cell membrane accompanying a change in the ion permeability of the nerve cell, and a change in the cell membrane potential accordingly. Therefore, the activity of the nerve cell can be detected by measuring the potential change accompanying the change in the ion concentration near the nerve cell (that is, the ionic current) using the electrodes.

The measurement of the potential due to the cell activity as described above can be performed, for example, by inserting a glass extracellular potential measuring electrode into the cell. When measuring the evoked potential due to stimulation, a metal electrode for stimulation is inserted together with a glass electrode for recording. However, the measurement by inserting these electrodes may damage the cells, and it is difficult to perform the measurement for a long time. Further, there are problems in space limitation and position accuracy in simultaneously measuring a plurality of points.

Therefore, the inventors of the present invention invented an integrated composite electrode in which a plurality of microelectrodes and their extraction patterns are formed on an insulating substrate by using a conductive substance, and cell culture can be performed on the microelectrodes. (JP-A-6-78889,
See JP-A-6-296595). If this integrated composite electrode is used, it is possible to measure the potential change at a plurality of places at narrow intervals without being restricted by space, and it is possible to measure the potential change for a long time.

[0005]

However, by making maximum use of such an integrated composite electrode, the measurement can be accurately and efficiently performed and the measurement results can be conveniently arranged. A device or measurement system was strongly desired. It is an object of the present invention to provide a cell potential measuring device that meets such a demand.

[0006]

The cell potential measuring device according to the present invention comprises: A plurality of microelectrodes are provided on a substrate, a cell setting portion for placing cells on the microelectrode is provided, and an electric connection means for applying an electric signal to the microelectrode and deriving an electric signal from the microelectrode is provided. An integrated cell setting device provided, and B. C. a stimulus signal providing means connected to the electrical connection means of the cell placement device for providing an electrical stimulus to the cells; Signal processing means connected to the electrical connection means of the cell placement device for processing an output signal due to the electrophysiological activity of the cell.

[0007] Preferably, an optical observation means for optically observing the cells is provided, and a cell culture means for maintaining a culture atmosphere of the cells placed on the integrated cell placement device is further provided. With the provision, measurement can be performed for a long time.

Other preferable configurations will be described later together with the operation.

[0009]

The outline of the measurement by the apparatus of the present invention having the above-mentioned structure is performed as follows, for example. A sample cell is set in the cell setting part of the integrated cell setting device, and a plurality of microelectrodes contact the cell. While referring to the image of the cell obtained by the optical observation means, the stimulation signal applying means applies the stimulation signal between an arbitrary pair of electrodes among the plurality of microelectrodes through the electrical connection means. The time change of the evoked potential obtained in each of the other electrodes is given to the signal processing means via the electrical connection means, and is output to, for example, a display device or the like through necessary signal processing. In addition, the measurement of the self-power generation position without giving a stimulation signal is performed in the same manner.

Since the electrochemical measurement of cells as described above needs to be carried out in a living state, normally, cultured cells are used, and the cell setting part of the integrated cell setting device is provided with a medium. There is. The integrated cell placement device is detachable from the measuring device, and the integrated cell placement device is placed in an ordinary incubator for cell culture, and the integrated cell placement device is taken out of the incubator for measurement. Although it may be set, if a cell culture means for maintaining the culture atmosphere of the cells on the integrated cell placement device is further provided, the measurement can be performed for a long time. This cell culture means is a temperature control means for keeping the temperature constant,
It is composed of means for circulating the culture solution and means for supplying a mixed gas of air and carbon dioxide (for example, CO ₂ 5%).

Preferably, the integrated cell placement device comprises a plurality of microelectrodes arranged in a matrix (lattice) on a glass plate, a conductive pattern for drawing out these microelectrodes, and a conductive pattern for these conductive electrodes. The cell mounting portion is provided in an area including the plurality of microelectrodes, the electrical contact being connected to the end portion, and the insulating coating covering the surface of the conductive pattern. Using a transparent glass plate as a substrate facilitates optical observation of cells. Therefore, it is preferable that the conductive pattern and the insulating coating are also substantially transparent or semi-transparent. Further, if a plurality of microelectrodes are arranged in a matrix, it is easy to specify the position of the electrode to which the stimulation signal is applied and the electrode to detect the voltage signal due to the cell activity. For example, it is preferable to arrange 64 micro electrodes in 8 columns and 8 rows. Further, the surface area of each electrode is preferably as wide as possible from the viewpoint of minimizing the surface resistance and increasing the detection sensitivity, but in consideration of the constraint from the electrode interval, the measurement resolution, etc., 4 × 10 ² to 4 × 10
It is preferably in the range of ⁴ μm ² .

[0012] It is preferable that the electric connecting means includes a two-divided holder that has a contact fitting for contacting the electric contact and that fixes the glass plate by sandwiching it from above and below,
With such a structure, the glass plate can be fixed and the microelectrodes can be drawn out easily and surely. Furthermore, it is preferable to further include a printed wiring board that fixes the holder and has an external connection pattern that is connected to the contact fittings of the holder via a connector, whereby an external device, that is, a stimulation signal providing unit and a signal. Connection with the processing means becomes easy. In order to transmit the stimulation signal and the detection signal without attenuation and distortion as much as possible, the contact resistance between the electrical contact and the contact fitting and the contact resistance between the contact fitting and the connector are preferably 30 milliohms or less. .

It is preferable that the optical observation means includes an optical microscope and an image pickup device and an image display device connected to the optical microscope. In other words, the image of the cell magnified by the microscope is captured by the image capturing device (video camera) and displayed on the image display device (high definition display), so that the measurement can be performed while visually recognizing the cell and electrode positions. It will be easy to do. More preferably, if the optical observation means is equipped with an image storage device, it is convenient for recording measurement results and the like.

Further, by using a pulse signal generator for the stimulation signal applying means, many kinds of signal waveforms can be applied to cells as stimulation signals. The signal processing means includes a multi-channel amplifier that amplifies a detection signal due to cell activity, and a multi-channel display device that displays the amplified signal waveform in real time, and a signal waveform (cell potential time) obtained from a plurality of electrodes. (Change) can be displayed simultaneously.

On the other hand, a computer is further provided which outputs the stimulus signal through the D / A converter and inputs and processes the output signal due to the electrophysiological activity of the cell through the A / D converter. It is preferable that the stimulus signal is set to an arbitrary waveform on the screen, the waveform of the detection signal is displayed on the screen as well as variously processed, displayed, output to a plotter, or stored. It becomes easy. Furthermore, the computer can be used to control the optical observation means and the cell culture means.

[0016]

Embodiments of the present invention will be described below with reference to the drawings.

First, the integrated cell setting device used in the present measuring apparatus will be described. As shown in a perspective view in FIG. 1 and an assembly view in FIG. 2, this integrated cell placement device 1 is an integrated composite electrode 2 in which a plurality of microelectrodes and their extraction patterns are provided on a glass plate. It is composed of two divided holders 3 and 4 which are sandwiched and fixed from above and below, and a printed wiring board 5 which fixes the holders.

The integrated composite electrode is described in JP-A-6-7.
This is almost the same as that disclosed in Japanese Patent No. 8889. 64 microelectrodes 11 in the center of a substrate made of transparent Pyrex glass with a thickness of 1.1 mm and a size of 50 mm
Are formed in an 8 × 8 matrix, and a conductive pattern 12 for extraction is connected to each microelectrode (see FIG. 3). Each electrode 11 is 50 μm square (area 25 × 10 ² μ
m ² ), and the distance between the centers of adjacent electrodes is 450 μm
Is. Further, 16 electric contacts 7 are formed on each of the four sides of the substrate, 64 in total (see FIG. 2), and these electric contacts correspond to the 64 microelectrodes in the central portion of the substrate in a one-to-one correspondence. Thus, the conductive patterns for extraction 12 are connected. The 16 electrical contacts on each side are arranged at a pitch of 1.27 mm. A method of manufacturing the integrated composite electrode 2 will be described below with reference to the sectional view of FIG. Note that FIG. 4 is drawn by changing the scale of each part for ease of viewing.

An ITO (indium tin oxide) film having a thickness of 150 nm is applied on the surface of the glass plate 13, and a conductive pattern 12 is formed by photoresist and etching. A 1.4 .mu.m thick negative photosensitive polyimide film is applied thereon, and an insulating film 14 is formed in the same manner. The ITO film is exposed at the portions of the microelectrodes and the electrical contacts, and nickel plating with a thickness of 500 nm is formed on this portion.
And gold plating 16 with a thickness of 50 nm is applied. Inner diameter 22 mm,
Cylindrical polystyrene frame 6 with an outer diameter of 26 mm and a height of 8 mm
(See FIG. 2) is bonded to a glass plate (via the conductive pattern 8 and the insulating coating 9) using a silicone-based adhesive. This cylindrical polystyrene frame is fixed in a state where it is aligned with the center of the glass plate, that is, the central parts of the 64 microelectrodes, and the inside of this polystyrene frame corresponds to the cell setting part. 1 wt% chloroplatinic acid, 0.01 wt% lead acetate, 0.0025 wt% in this polystyrene frame
Platinum black 11a is deposited on the surface of the gold plating of the microelectrode portion by filling the aqueous solution of hydrochloric acid and applying a current of 20 mA / cm ² for 1 minute.

Next, the two-piece holders 3 and 4 for sandwiching and fixing the integrated composite electrode 2 from above and below will be described. The holders 3 and 4 are made of resin, and as shown in FIG. 2, a step portion for holding the edge portion of the integrated composite electrode 2 and a rectangular opening are provided in the central portion. The upper holder 3 is provided with a pair of fixtures 8 and 16 × 4 pairs of contact fittings 9. FIG. 5 (A) is a top view of the holders 3 and 4 fixed with the integrated composite electrode 2 sandwiched therebetween, and FIG. 5 is a side view thereof (cross-sectional view taken along line BB).
FIG. 6 shows a perspective view seen from (B) and from the back side, respectively. As can be seen from these figures, the fixture 8 is pivotally supported by the shaft pins 8a on the two opposite sides of the upper holder 3. Grooves 4a are formed on the opposite two sides of the back surface of the lower holder 4, and the protrusions 8b of the fixture 8 are fitted therein so that the upper and lower holders 3 and 4 sandwich the integrated composite electrode 2 therebetween. It is firmly fixed in the state.

The 64 contact fittings 9 provided on the upper holder 3 corresponding to the electric contacts 7 of the integrated composite electrode 2 are
It is formed by processing a metal plate with good elasticity such as BeCu plated with Ni and Au, and has a shape as shown in FIG. That is, it is composed of the pin portion 9a and its base portion 9b, and the movable contact portion 9d extending from this base portion 9b via the curved portion 9c. With such a structure, the movable contact portion 9d can be elastically displaced with respect to the base portion 9b. The upper holder 3 is formed with holes (64 × 16) where the pin 9a of the contact fitting 9 is inserted and the groove into which the base 9b is fitted.

As shown in FIGS. 2 and 5B, the pin 9a projects from the upper holder 3 with the contact fitting 9 inserted and fixed in the hole and groove. By alternately arranging two types of contact fittings 9 having different lengths of the base portion 9b, 16 pin portions 9a protruding from the upper holder 3 are formed.
Are arranged in two staggered rows. As will be described later, these pin portions 9a are connected to a connector mounted on the printed wiring board 5 for external connection.

On the other hand, the movable contact portion 9d of the contact fitting 9 projects from the lower surface of the upper holder 3 with the contact fitting 9 inserted and fixed in the hole and groove of the upper holder 3. This state is well shown in FIG. 8 which is an assembly view seen from the opposite side to the assembly view of FIG. With the holders 3 and 4 fixed with the integrated composite electrode 2 sandwiched therebetween, the movable contact portion 9d of each contact fitting 9 comes into contact with the electrical contact 7 of the integrated composite electrode 2, and the curved portion 9c is elastically deformed to a predetermined extent. The contact pressure of is applied to the contact part. In this way, the integrated composite electrode 2
The electrical contact 7 that is connected to the microelectrode 11 of 11 through the conductive pattern 12 has a small contact resistance (30
Below the million)) electrically connected.

Next, the printed wiring board 5 will be described. This printed wiring board 5 fixes the assembly of the integrated composite electrode 2 and the holders 3 and 4, and extends from the microelectrode 11 of the integrated composite electrode 2 to the conductive pattern 12, the electrical contact 7, and the contact fitting 9. It is also responsible for drawing electrical connections out through the connector. In addition, it also has the function of facilitating the handling such as setting on the measuring device.

This printed wiring board 5 is constructed by using a glass epoxy substrate having a double-sided pattern, and on the back surface shown in FIG. 8, connectors 5a are provided at four locations around a circular opening formed in the central portion. ing. Upper holder 3
By inserting the 16 pin portions 9a protruding in a zigzag in two rows from the four surfaces of the connector into the corresponding connectors 5a, the assembly of the integrated composite electrode 2 and the holders 3 and 4 forms a printed wiring board 5. It is fixed to and electrically connected.

Electrical contacts of 2.54 mm pitch for double-sided edge connectors are formed on both side edge portions 5b of the printed wiring board 5, and these electrical contacts and the central connector 5a are formed.
And are connected by a drawing pattern 5c. The inner row of the double-sided connector 5a is drawn out as a front surface pattern and the outer row is drawn out as a back surface pattern, and 32 electric contacts are formed on each of the edge portions 5b on both front and back surfaces, so that a total of 64 electric contacts are formed. In order to secure the mechanical fixation, the upper holder 3 is fixed to the printed wiring board 5 with screws.
It can also be fixed to.

FIG. 9 shows a preferred embodiment of a cell potential measuring device constructed by using the integrated cell placement device 1 constructed as described above. The measuring apparatus of the present embodiment includes the above-mentioned integrated cell placement device 1, an optical observation means 20 including an inverted microscope 21 for optically observing the cells set in the integrated cell placement device 1, and a cell. A computer 30 including a means for applying a stimulus signal to the cell and a means for processing an output signal from the cell, and a cell culture means 40 for maintaining a cell culture atmosphere.

The optical observation means 20 has an inverted microscope 21 (IMT manufactured by Olympus) in which the integrated cell setting device 1 is set.
-2-F or IX70 equivalent), SI for microscope
T camera (equivalent to C2400-08 manufactured by Hamamatsu Photonics)
22, high-definition display 23, and image file device (TQ-2600 or FTQ-3100F manufactured by Matsushita Electric Industrial Co., Ltd.)
Equivalent) 24 is included. However, the high definition display may also serve as the display of the computer 30. Note that the specific devices shown in the above parentheses are examples, and the present invention is not limited to these. The same applies hereinafter.

As the computer 30, a WINDOWS compatible personal computer equipped with an A / D conversion board and measurement software is used. A / D
The conversion board is the A / D converter 31 and the D / A converter 3 shown in FIG.
Includes 2. The A / D converter 31 has 16 bits and 64 channels, and the D / A converter 32 has 16 bits and 8 channels.

The measurement software includes software for setting conditions for applying a stimulus signal and recording conditions for the obtained detection signal. With this measurement software, the computer 30 not only constitutes a means for giving a stimulus signal to cells and a means for processing signals detected from cells, but also an optical observation means (SIT camera and image file device), It also controls cell culture means. less than,
The main specifications of the measurement software are explained for each screen.

On the parameter setting screen, complicated stimulation conditions can be set by drawing a stimulation waveform on the screen using a keyboard or a mouse. The recording conditions are set with 64 input channels and 10 sampling rates.
It is designed to support continuous recording for several hours at kHz. Further, regarding the designation of the electrode that gives the stimulation signal and the electrode that takes out the detection signal from the cell, the microscope image displayed on the screen can be designated by using a mouse or a pen. In addition, various conditions such as temperature and pH of the cell culture means 40 can be set using a keyboard.

On the recording screen, spontaneous action potentials or induced potentials detected from cells are displayed in real time on a maximum of 64 channels. In addition, the recorded spontaneous action potential or induction potential can be displayed by being superimposed on the microscopic image of the cell. When measuring evoked potentials, the entire recorded waveform is displayed. In the case of spontaneous action potential measurement, the recorded waveform is displayed only when the occurrence of spontaneous action is detected by the spike detection function using the window discriminator or the waveform discriminator. Along with the display of the recording waveform, the measurement parameters (stimulation condition, recording condition, temperature, pH, etc.) at the time of recording are also displayed in real time. An alarm function is also provided when the temperature or pH is out of the allowable range.

On the data analysis screen, FFT analysis, coherence analysis and correlation analysis are possible. Single spike separation function using waveform discriminator,
It also has a temporal profile display function, a topography display function, and a current source density analysis function. These analysis results can be displayed by being superimposed on the microscope image stored in the image file device.

When the stimulus signal is output from the computer 30 as described above, the stimulus signal is output to the D / A converter 32.
And an isolator (equivalent to BSI-2 manufactured by BAK ELECTRONICS) 33 to the cells. That is, the stimulation signal is applied between the selected two points of the 64 microelectrodes 11 of the integrated cell placement device 1. And each microelectrode 1
The evoked potential generated between 1 and the GND level (potential of the culture solution) is a high-sensitivity amplifier for 64 channels (Nihon Kohden A.
B-610J equivalent) 34 and A / D converter 31 and input to the computer 30. The amplification factor of the amplifier 34 is 100 dB, and the frequency band is 0 to 10 kHz. However, in the case of measuring the induced potential by the stimulation signal, the frequency band of 100 Hz to 10 kHz is set by the low cut filter.

Next, the cell culture means 40 is provided with a temperature controller 41, a culture fluid circulation means 42, and a means 43 for supplying a mixed gas of air and carbon dioxide. Actually, Medical Systems Micro Incubator PDMI-2 equivalent and temperature controller TC-202
The cell culturing means 40 is configured by using an equivalent product and a CO ₂ cylinder or the like. This microincubator can be controlled in a temperature range of 0 to 50 ° C. by a Peltier device, and can correspond to a liquid feed rate of 3.0 ml / min or less and an air supply rate of 1.0 l / min or less. Alternatively, a microincubator (IMT2-IBSV manufactured by Olympus Corporation) having a built-in temperature controller may be used.

The preferred embodiment of the cell potential measuring device according to the present invention has been described above. However, the cell potential measuring device according to the present invention is not limited to the above embodiment, and various modifications such as those described below are possible. It can be implemented.

In the above-mentioned embodiment, the means for giving the stimulus signal to the cell is constituted by the computer and the D / A converter, but it may be constituted by a general-purpose or dedicated pulse signal generator. still,
The stimulus signal is to remove the effects of artifacts:
In order to prevent the DC component from flowing, it is preferable to use a bipolar constant voltage pulse composed of a pair of positive and negative pulses. Further, it is preferable to convert to a constant current pulse so that an overcurrent does not flow. For example, pulse width 100 μsec
It is preferable that the stimulus signal is composed of the positive pulse of, the interval of 100 μsec, and the negative pulse of 100 μsec, and the peak current of the positive and negative pulses is in the range of 30 to 200 μA.

Further, since the measuring device is equipped with the cell culture means 40, continuous measurement can be performed for a long period of time. However, the sample cells are integrated into an integrated cell setting device in an incubator installed separately from the measuring device. Alternatively, the integrated cell placement device may be taken out of the incubator and set in the measurement device only when the measurement is performed for a relatively short time. In this case, the cell culture means 40 need not be provided in the measuring device.

Using the above-described cell potential measuring device,
An example of actually measuring the potential change associated with the activity of nerve cells or tissues cultured on the integrated cell placement device will be described below. Using a section of rat cerebral cortex as neural tissue,
Culture was carried out by the method shown in the culture method example described later.

First, the result of comparison between the voltage waveform measured using the integrated cell placement device of this device and the voltage waveform measured using the conventional general-purpose glass electrode (extracellular potential measurement electrode) will be described. I'll do it. Measurement is 14 after the start of culture
The nerve tissue that had passed the day was used as a sample. A stimulus signal was applied between two adjacent electrodes of the integrated composite electrode constituting the integrated cell placement device, and the waveform of the time-dependent change in the evoked potential evoked at the eight electrodes near the electrode was measured. Then, for comparison, a glass electrode was sequentially moved to the vicinity of the above eight electrodes using a three-dimensional micromanipulator and the same voltage waveform was measured.

As described above, as a result of comparing the voltage waveform measured using the integrated composite electrode (integrated cell placement device) at eight locations with the waveform measured using the glass electrode, both waveforms were The location was also very similar.
Typical examples thereof are shown in FIGS. 10 (A) and 10 (B). Figure 10
FIG. 10A shows a waveform measured by the integrated composite electrode.
(B) shows a waveform measured by a glass electrode. Comparing the two waveforms, it can be seen that there is a slight difference in the frequency characteristics. The measurement using the glass electrode is slightly impaired in the ability to follow a rapid potential change, as compared with the measurement using the integrated composite electrode (planar electrode). It is considered that this difference is due to the difference in electric capacity between the glass electrode and the planar electrode.

Next, the experimental results of investigating the relationship between the number of days of culturing nerve cells cultured on the integrated cell placement device and the distribution of potential due to cell activity will be described. Prior to culturing the cells, the surface of the integrated composite electrode was covered with collagen gel for the purpose of enhancing the adhesiveness between each electrode of the integrated composite electrode and the cells. That is, as described above, a collagen gel having a thickness of 50 μm or less was formed on the surface of each electrode coated with platinum black and the surface of the insulating coating around it. Then, a section of rat cerebral cortex (thickness of 500 μm or less) was placed on the collagen gel and positioned on the microelectrode and cultured. FIG. 11 shows the measurement result of the self-power generation position, and FIG. 12 shows the measurement result of the evoked potential when the stimulation signal is given.

FIG. 11 (A) shows a microscope image of the sample cells and microelectrodes, and the waveforms of the self-power generation positions measured at the seven electrodes indicated by 1 to 7 are respectively shown in this image. 11 (B) and (C). FIG. 11 (B)
Is a waveform on the 6th day after culturing, and FIG.
This is the waveform on day 0. The scale of the microscope image, the time and voltage scale of the measurement waveform are as shown in the figure. From the measurement results, for example, on the 6th day after the culture, the spontaneous activity of the cells measured at each electrode is weak and the synchrony between the electrodes is hardly seen, whereas on the 10th day after the culture, a large number of nerve cells are detected. It can be seen that the electrodes are activated at the same time and the synchronism between the electrodes is increased.

FIG. 12A also shows microscopic images of sample cells and microelectrodes. Then, by the image processing included in the measurement software of the computer described above, the contour of the cell and the position of each electrode were drawn from this microscope image on the screen, and the voltage waveform measured at each electrode was displayed in an overlapping manner. Things are displayed in FIGS. 12 (B) and 12 (C). FIG. 12 (B) shows the evoked potential distribution on the 5th day after culturing, and FIG. 12 (C) shows the evoked potential distribution on the 10th day after culturing. The pair of electrodes indicated by + and-in the upper right is the electrode to which the stimulation signal is applied. Immediately above the small square symbol indicating the position of each electrode, the waveform measured at that electrode is displayed.
In these waveforms, the portion of the left end that largely shakes vertically is an artifact that directly corresponds to the stimulation signal,
Subsequent changes in potential indicate actual cell activity.
From the measurement results, for example, on the 5th day after the culturing, the cell activity was limited to the relatively close position of the electrode to which the stimulation signal was applied, but on the 10th day after the culturing, the cell activity was observed over a wide range. It can be seen that the size (amplitude) becomes large. [Example of cerebral cortex culture method] (1) Medium 1: 1 of Dulbecco's modified Eagle medium and Ham's F12 medium
The following additives were added to a mixed medium (GIBCO Co., LTD. 430-2500EB) and used. * Glucose (GIBCO 820-5023IN) 2.85mg / L (total 6mg / L including the one originally contained in the above medium) * Putrescine (putrescine, SIGMA Co., LTD. P5780) 10
0 μM * Progesterone (progesterone, SIGMA P8783) 20nM * Hydrocortisone (hydrocortisone, SIGMA H0888)
20nM * Sodium selenite (sodium selenite, WAKO Co., L
TD. 198-0319) 20nM * Insulin (insulin, SIGMA 16634) 5mg / L * Transferrin (transferrin, SIGMA T1147) 100mg /
L * Sodium bicarbonate 2.438mg / L * Adjust the pH to 7.4 by adding an appropriate amount of 1N HCl or 1N NaOH.

After the above additives were added and sterilized by filtration, 4 ° C
Saved in and ready for use. Hereinafter, this medium is simply referred to as "medium"
Call. (2) Construction of wells on integrated composite electrode For culturing nerve cells or nerve tissue on integrated composite electrode
For inner diameter, 22mm inner diameter, 26mm outer diameter, 8mm height police
The cylinder made of Tiren was adhered by the method described below. (A) Polystyrene cylinder (inner diameter 22 mm, outer diameter 26 mm, height
8mm) on the bottom surface of 1-component silicone adhesive (Dow Corn
891 or Shin-Etsu Chemical KE-42RTV)
It (B) Glass substrate center of integrated composite electrode and polystyrene
Bond the two, making sure that the centers of the cylinders are aligned.
To do. (C) By leaving it for 24 hours in an environment where dust cannot enter
Solidify the adhesive. (D) Inside a clean bench after soaking in 70% ethanol for 5 minutes
For sterilization by air-drying with
Prepare (3) Treatment of electrode surface In order to enhance cell adhesion of the integrated composite electrode surface,
A collagen gel was constructed on the electrode surface by the method. The following operations
All the works were performed under a sterile atmosphere. (A) Prepare the following solutions A, B, and C, and cool them with ice. A. 0.3 vol.% Dilute hydrochloric acid collagen solution (pH 3.0, Nitta gelatin
Cellmatrix Type I-A) B. Double Becco's modified Eagle's solution and Ham's F12 medium
Sodium bicarbonate in 1: 1 mixed medium (GIBCO 430-2500EB)
Without adding, make a 10 times more concentrated solution than normal use,
Filter sterilized C.I. Bicarbonic acid is added to 100 mL of 0.05N sodium hydroxide solution.
Dissolve 2.2g of sodium and 4.77g of HEPES (GIBCO 845-1344IM)
And sterilized by filtration (b) Cooling solution A, B, C at a ratio of 8: 1: 1
Mix. At this time, add C well after mixing A and B well.
Well, mix further. (C) Integrated composite that has been cooled to approximately 4 ° C in advance
Dispense 1 mL of the mixed solution of b) into the well of the electrode and
After covering the pole surface thoroughly,
Remove as much mixed solution as possible with your pet. This operation
A coating of a mixing liquid with a thickness of 50 μm or less on the electrode surface.
Be composed. (D) The integrated composite electrode with the mixed liquid coating is placed at 37 ° C.
Warm for 30 minutes to gel the mixing solution,
It constitutes the collagen matrix. (E) Add 1 mL of sterilized water to the well of the integrated composite electrode.
Yes, leave it for about 5 minutes and then remove it for cleaning.
U The operations of (f) and (e) are repeated two more times (three times in total). (G) In the well of the integrated composite electrode, the modified Dulbecco's
Eagle's medium and Ham's FH12 medium 1: 1 mixed medium (GIBC
O 430-2500EB) with the above additives added (however,
Dispense 1 mL (excluding insulin and transferrin)
Temperature 37 ° C, relative humidity 97% or more, CO₂Concentration 5
%, CO kept at 95% air concentration₂In the incubator
Save it and prepare for use. (4) Culturing of nerve cells or nerve tissues Culture forms are roughly classified into two types. That is, God
It is a dispersion culture of transcells and an organ culture of neural tissue. Less than,
Each will be described. (4-1) Dispersion Culture Method of Rat Cerebral Cortex Visual Cortex Neurons The following operations were all performed in a fungal atmosphere. (B) SD rat fetus 16-18 days after pregnancy
Brains were removed and ice-cold Hanks balanced salt solution (GIBCO 450-1
250 EB). (B) Cutting the visual cortex from the brain in ice-cold Hanks balanced salt solution
And transfer to the minimum essential medium of torr (GIBCO 410-1100EB).
You (C) The visual cortex can be formed in Eagle's minimum essential medium solution
Only cut finely to a maximum of 0.2 mm square
It (D) Insert the finely cut visual cortex into a centrifuge test tube.
And calcium- and magnesium-free Hanks
After washing three times with a balanced salt solution, the solution is dispersed in an appropriate amount of the same solution. (E) 0.25% in the test tube for centrifugation in (d) above.
Calcium and magnesium with dissolved trypsin
Double the total volume by adding Hanks Balanced Salt Solution without it. Loose
Keep constant temperature at 37 ℃ for 15 minutes with gentle stirring.
Then, the enzyme reaction is performed. (F) The medium described in (1-1) above (including additives.
Below, "abbreviated as" medium "), 10 vol.% Fetal bovine blood
Add the liquid to a test tube for centrifugation through the above e).
Add in the medium and double the total volume. Burner the tip
Use a glass Pasteur pipette with a small diameter to loosen
Repeat pipetting gently (up to 20 times),
Individual cells are isolated. (G) 9806.65 m / sec²(Ie 1000
g) was centrifuged for 5 minutes. Supernatant after centrifugation
And discard the pellet and suspend the pellet in a medium containing 5% fetal calf serum.
It (H) The above (g) is repeated two more times (three times in total). (I) The final obtained precipitate was treated with 5 vol.% Fetal bovine serum.
Suspend in a medium containing and count the cell concentration in the suspension with red blood cells
Measure using a plate. After measurement, use the same medium to
Concentration is 2-4 × 10⁶Adjust so that the number becomes 1 / ml. (E) CO after the treatment of (1-3) above₂Incube
Remove the integrated composite electrode stored in the
Medium in Ell (but insulin and transfer
(Phosphorus-free) was removed, and a new 5 vol.% Fetal calf
Dispense 500 μL of medium containing serum. In addition,
Gently add 100 μL of the cell suspension after adjusting the cell concentration,
CO again₂Place in incubator. (L) After 3 days from the operation in (u) above, add half of the culture medium
Replace with a new one. Medium to be replaced contains fetal bovine serum
No medium was used. To reduce the concentration of fetal calf serum
Therefore, the number of cells other than nerve cells (for example, clear cells) increases.
Suppress breeding. (W) After that, the medium is replaced in the same manner as above every 1 to 2 days. (4-2) Rat cerebral cortex slice culture method (a) The brain was taken out from the SD rat on the 2nd day after birth and was frozen on ice.
Hanks equilibrium with cold 0.25 vol.% D-glucose
Soak in salt solution. (B) Ice-cooled HB containing 0.25 vol.% D-glucose
In SS, the brain membrane attached to the brain is damaged in the cerebral cortex.
Use sharp tweezers, being careful not to
Excluded. (C) 500μ from the corpus callosum on one side of the cerebral cortex excluding the cerebral membrane
Using micro scissors for eye surgery at about m,
Cut along the corpus callosum from the occipital side toward the frontal side
It (D) Then, using micro scissors for eye surgery,
Large with a thickness of 200-300 μm perpendicular to the cut surface of (C)
Brain cortex is cut and sectioned. (E) Furthermore, using micro scissors for ophthalmic surgery, section
Adjust the size to about 1 × 1. (F) Prepare in the above “(3) Treatment of electrode surface”
Integrated composite electrode with CO ₂Remove from incubator
And adjust the size of the cerebral cortex slice with a diameter of 2 mm or more.
Gently suck it up without damaging it with a pipette, and
Transfer to the culture well of the combined electrode. (G) Pasteur with a smooth burner tip
Be careful not to damage the cerebral cortex section with a pipette
While the cortical layer structure faces the upper surface and is located on the electrode
Adjust to (H) After placing the cerebral cortex section on the integrated composite electrode,
Adjust the amount of ground, the bottom of the section touches the culture medium, the top is outside air
To touch. (I) After adjusting the amount of medium, attach the integrated composite electrode to a sterile petri dish.
Approximately 5m of sterile water at 37 ℃ to prevent the medium from drying
Dispense 1 into a Petri dish and add CO again₂In the incubator
Let stand. After that, pay attention to the amount of medium and change the medium once a day.
went. The amount of medium is the same as in (h) above.
It

[0046]

As described above, according to the cell potential measuring apparatus of the present invention, the activity of living cells set in the integrated cell placement device can be simultaneously measured at a plurality of locations without being damaged. The electric potential can be measured. And
Not only can the self-power generation position be measured from any of a plurality of electrodes, but the evoked potential obtained by applying a stimulus between any of the electrodes can also be measured simultaneously from a plurality of electrodes.
Further, by processing using a computer or the like, for example, an electrode to which a stimulation signal is given while confirming the relationship between the sample cell and the electrode position from the microscope image obtained by the optical observation means, or the voltage waveform obtained from each electrode It is also possible to display the cells on the image showing the cell and electrode positions,
It can greatly contribute to making experiments in the field of electroneurophysiology efficient and accurate.

Further, by providing the cell culture means for maintaining the culture atmosphere of the cells set in the integrated cell placement device, the continuous measurement for a long period can be stably performed while the sample cells are set in the measuring device. be able to.

[Brief description of drawings]

FIG. 1 is a perspective view of an integrated cell placement device used in a cell potential measuring device according to an embodiment of the present invention.

[Fig. 2] Assembly drawing of integrated cell placement device

FIG. 3 is a plan view showing 64 microelectrodes and extraction patterns provided in the central portion of an integrated composite electrode which constitutes an integrated cell placement device.

FIG. 4 is a diagram schematically showing a cross section of an integrated composite electrode.

FIG. 5 is a plan view and a side sectional view showing a state in which the integrated composite electrode is sandwiched and fixed by the upper and lower holders.

6 is a perspective view of the integrated composite electrode and the upper and lower holders of FIG.

FIG. 7 is a side view of a contact fitting provided on the upper holder

FIG. 8 is an assembly view of the integrated cell placement device viewed from the opposite direction to FIG.

FIG. 9 is a block configuration diagram of a cell potential measuring device according to an embodiment of the present invention.

FIG. 10 is a voltage waveform based on the activity of cultured cells measured using the integrated cell placement device used in the device of the present invention and a conventional general-purpose glass electrode (extracellular potential measurement electrode). Diagram showing an example of comparison with voltage waveforms

FIG. 11 is a diagram showing the measurement result of the self-power generation position of cultured cells measured using the device of the present invention.

FIG. 12 is a diagram showing the measurement results of evoked potentials of cultured cells measured using the device of the present invention.

[Explanation of symbols]

 1 Integrated Cell Installation Device 2 Integrated Composite Electrode 3 Upper Holder 4 Lower Holder 5 Printed Wiring Board 11 Microelectrode 12 Conductive Pattern 20 Optical Observation Means 21 Inverted Microscope 22 SIT Camera 24 Image Storage Device 30 Computer 31 A / D Converter 32 D / A converter 33 Isolator 34 Multi-channel amplifier 40 Cell culture means

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display location G01N 27/28 341 Z 27/416 33/483 E (72) Inventor Makoto Takeya Osaka Kadoma City, Osaka 1006 Kadoma Matsushita Electric Industrial Co., Ltd. (72) Inventor Tadayasu Kouji 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd.

Claims (15)

[Claims] 1. A cell potential measuring device for measuring electrophysiological properties of cells, comprising: A plurality of microelectrodes are provided on a substrate, a cell setting portion for placing cells on the microelectrode is provided, and an electric connection means for applying an electric signal to the microelectrode and deriving an electric signal from the microelectrode is provided. An integrated cell setting device provided, and B. C. a stimulation signal providing means connected to an electrical connection means of the integrated cell placement device for providing an electrical stimulation to the cells; A signal processing unit connected to an electric connection unit of the cell placement device for processing an output signal due to the electrophysiological activity of the cell. 2. The cell potential measuring device according to claim 1, further comprising an optical observation means for optically observing the cells. 3. The cell potential measuring device according to claim 1, further comprising cell culture means for maintaining a culture atmosphere of cells placed on the integrated cell placement device. 4. The cell potential measurement according to claim 3, wherein the cell culture means comprises a temperature control means for keeping the temperature constant, a means for circulating the culture solution, and a means for supplying a mixed gas of air and carbon dioxide. apparatus. 5. The integrated cell placement device comprises a plurality of microelectrodes arranged in a matrix on a glass plate,
A conductive pattern for drawing out these microelectrodes, an electrical contact connected to an end of these conductive patterns, and an insulating film covering the surface of the conductive pattern are provided, and the cells are provided in an area including the plurality of microelectrodes. The cell potential measuring device according to claim 1, further comprising an installation portion. 6. The cell potential measuring device according to claim 1, wherein 64 electrodes are arranged in 8 columns and 8 rows as the plurality of micro electrodes. 7. The electrode area of each of the microelectrodes is 4
The cell potential measuring device according to claim 1, which is in the range of × 10 ² to 4 × 10 ⁴ µm ² . 8. The cell potential measuring device according to claim 5, wherein the electrical connecting means includes a two-divided holder which has a contact fitting for contacting the electrical contact and which holds the glass plate by sandwiching the glass plate from above and below. 9. The cell according to claim 5, wherein the electrical connection means further comprises a printed wiring board having an external connection pattern for fixing the holder and connecting to a contact fitting of the holder via a connector. Potential measuring device. 10. The cell potential measuring device according to claim 8, wherein a contact resistance between the electrical contact and the contact fitting and a contact resistance between the contact fitting and the connector are 30 milliohms or less, respectively. 11. The optical observation means comprises an optical microscope,
The cell potential measuring device according to claim 1, further comprising an imaging device and an image display device connected to the device. 12. The cell potential measuring device according to claim 10, wherein the optical observation means further includes an image storage device. 13. The cell potential measuring device according to claim 1, wherein the stimulation signal applying means is a pulse signal generator. 14. The signal processing means comprises a multi-channel amplifier for amplifying an output signal due to electrophysiological activity of the cell, and a multi-channel display device for displaying the amplified signal waveform in real time. Cell potential measuring device. 15. The stimulus signal is output via a D / A converter, and the output signal by the electrophysiological activity of the cells is input and processed via the A / D converter, and the optical observation is performed. The cell potential measuring device according to claim 1, further comprising a computer that controls the device and the cell culture device.