JP3204875B2 - Cell potential measurement device - Google Patents

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 electrical neurophysiology for measuring a potential change accompanying the activity of a nerve cell.

[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. When a nerve cell is activated, an action potential is generated.
This action potential is caused by a change in ion concentration inside and outside the cell membrane with a change in ion permeability of a nerve cell, and a change in the cell membrane potential with the change. Therefore, the activity of the nerve cell can be detected by measuring the potential change accompanying the ion concentration change (that is, the ion current) near the nerve cell by the electrode.

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 insertion of these electrodes may damage cells, and it is difficult to perform measurement over a long period of time. In addition, there is a spatial limitation and a problem of positional accuracy in simultaneously measuring a plurality of locations.

Accordingly, the present inventors have invented an integrated composite electrode in which a plurality of microelectrodes and a lead pattern thereof are formed on an insulating substrate by using a conductive substance, and cell culture can be performed thereon. (Japanese Patent Laid-Open No. 6-78889,
See JP-A-6-296595). By using this integrated composite electrode, the potential change can be measured simultaneously at a plurality of locations at a narrow interval without being restricted by space, and can be measured over a long period of time.

[0005]

However, by making full use of such an integrated composite electrode, the measurement can be performed accurately and efficiently and the measurement results can be conveniently arranged. There was a strong need for a device or measurement system. An object of the present invention is to provide a cell potential measuring device that meets such a demand.

[0006]

SUMMARY OF THE INVENTION A cell potential measuring device according to the present invention is a cell potential measuring device for measuring the electrophysiological properties of cells, comprising: A plurality of microelectrodes are provided on a substrate, and a cell mounting portion for mounting cells on the microelectrodes is further provided .
Connected to the conductive pattern and the end of the conductive pattern
B. an integrated composite electrode with a defined electrical contact; Electrical connection hands stage for deriving an electrical signal from the integrated multiple electrode while applying an electrical signal to the integrated multiple electrode, C. A stimulus signal application means connected to said electric connection means B for applying an electric stimulus to said cells; B. a signal processing means connected to said electrical connection means for processing an output signal from the electrophysiological activity of said cells ; The electrical connection means is a contact that contacts the electrical contact.
Hold the glass plate from above and below and fix it
A two-piece holder .

[0007] Preferably, by the cells comprising an optical observation means for optically observed, further example Bei cell culture means for maintaining a culture atmosphere of the cells, can be measured over time and that Do not.

[0008] Other preferred 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-described configuration is performed, for example, as follows. Integrated composite
A cell, which is a sample, is set in a cell setting portion of an integrated cell setting device provided with an electrode, and a plurality of microelectrodes contact the cell. A stimulus signal is applied between an arbitrary pair of the plurality of microelectrodes by the stimulus signal applying means via the electrical connection means while referring to the cell image obtained by the optical observation means. The time change of the evoked potential obtained for 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. Note that the measurement of the self-generated power level without giving a stimulus signal is performed in the same manner.

[0010] Since the electrochemical measurement of cells as described above needs to be performed while the cells are alive, cultured cells are usually used, and the cell installation section of the integrated cell installation device is provided with a medium. I have. The integrated cell setting device is detachable from the measurement device, and the entire cell setting device is placed in a normal incubator for cell culture, and when measuring, the integrated cell setting device is removed from the incubator and transferred to the measurement device. It may be set, but when a cell culture means for maintaining the culture atmosphere of the cells on the integrated cell setting device is further provided, measurement over a long period of time becomes possible. This cell culture means is a temperature control means for keeping the temperature constant,
It comprises a means for circulating a culture solution and a means for supplying a mixed gas of air and carbon dioxide (for example, 5% of CO ₂ ).

Preferably, the integrated cell setting device includes a plurality of microelectrodes arranged in a matrix (lattice) on a glass plate, a conductive pattern for leading out the microelectrodes, and a conductive pattern of these conductive patterns. An electrical contact connected to an end and an insulating coating covering the surface of the conductive pattern are provided, and the cell placement section is provided in a region including the plurality of microelectrodes. 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 translucent. In addition, if a plurality of microelectrodes are arranged in a matrix, it is easy to specify the position of an electrode for applying a stimulus signal or an electrode for detecting a voltage signal due to cell activity. For example, it is preferable to arrange 64 microelectrodes in 8 columns and 8 rows. The surface area of each electrode is preferably as large as possible from the viewpoint of reducing the surface resistance as much as possible and increasing the detection sensitivity. However, considering the restrictions from the electrode spacing and the measurement resolution, the surface area of each electrode is from 4 × 10 ² to 4 ×. 10
It is preferably in the range of ⁴ μm ² .

It is preferable that the electric connection means includes a two-piece holder having a contact fitting for contacting the electric contact and fixing the glass plate from above and below.
With this structure, the glass plate can be fixed and the microelectrodes can be easily and reliably drawn out. Further, it is preferable that the apparatus 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, whereby an external device, that is, a stimulus signal applying unit and a signal are provided. Connection with the processing means becomes easy. In order to transmit the stimulus signal and the detection signal as little as possible with attenuation and distortion, it is preferable that the contact resistance between the electrical contact and the contact fitting and the contact resistance between the contact fitting and the connector are each 30 mOhm or less. .

It is preferable that the optical observation means includes an optical microscope, and an imaging device and an image display device connected to the optical microscope. In other words, the image of the cell magnified by the microscope is picked up by the image pickup device (video camera) and displayed on the image display device (high-definition display), so that the measurement can be performed while visually grasping the cell and electrode positions. It is easier to do. More preferably, if the optical observation means has an image storage device, it is convenient for recording measurement results and the like.

By using a pulse signal generator as the stimulus signal applying means, many types of signal waveforms can be applied to cells as stimulus signals. The signal processing means includes a multi-channel amplifier for amplifying a detection signal due to cell activity, and a multi-channel display device for displaying 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, there is further provided a computer for outputting the stimulus signal via a D / A converter and for inputting and processing an output signal based on the electrophysiological activity of the cell via an 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 subjected to various processings, and output to the 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, an integrated cell setting device used in the present measuring device will be described. As shown in a perspective view in FIG. 1 and an assembly view in FIG. 2, the integrated cell placement device 1 is an integrated composite electrode 2 in which a plurality of microelectrodes and a drawing pattern thereof are provided on a glass plate. It consists of two divided holders 3 and 4 which are fixed from above and below, and a printed wiring board 5 which fixes this holder.

Regarding the integrated composite electrode, see JP-A-6-7
This is almost the same as that disclosed in Japanese Patent No. 8889 or the like. At the center of a substrate made of transparent Pyrex glass having a thickness of 1.1 mm and a size of 50 mm square, 64 microelectrodes 11 are provided.
Are formed in a matrix of 8 × 8, and a lead-out conductive pattern 12 is connected to each microelectrode (see FIG. 3). Each electrode 11 is 50 μm square (area 25 × 10 ² μm).
m ² ), and the distance between the centers of adjacent electrodes is 450 μm
It is. On each of the four sides of the substrate, a total of 64 electrical contacts 7 are formed, each of which is 16 (see FIG. 2), and these electrical contacts correspond to the 64 microelectrodes at the center of the substrate in a one-to-one correspondence. As described above. The 16 electrical contacts on each side are arranged at a pitch of 1.27 mm. A method for manufacturing the integrated composite electrode 2 will be described below with reference to the cross-sectional view of FIG. FIG. 4 is drawn with the scale of each part changed for easy 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 negative photo-sensitive polyimide film having a thickness of 1.4 μm is applied thereon, and an insulating film 14 is similarly formed. The ITO film is exposed at the microelectrodes and electrical contact portions, and nickel plating 15 nm thick
And gold plating 16 having a thickness of 50 nm. 22mm inside diameter,
Cylindrical polystyrene frame 6 having an outer diameter of 26 mm and a height of 8 mm
(See FIG. 2) is bonded to the glass plate (via the conductive pattern 8 and the insulating coating 9) using a silicone-based adhesive. The cylindrical polystyrene frame is fixed so as to be centered on the center of the glass plate, that is, the center of the 64 microelectrodes, and the inside of the polystyrene frame corresponds to the cell installation portion. 1% by weight of chloroplatinic acid, 0.01% by weight of lead acetate, 0.0025% by weight in this polystyrene frame
Filling with an aqueous solution of hydrochloric acid and applying a current of 20 mA / cm ² for 1 minute deposit platinum black 11a on the gold-plated surface of the microelectrode.

Next, the two-part holders 3 and 4 for fixing the integrated composite electrode 2 by sandwiching it from above and below will be described. The holders 3 and 4 are made of a resin, and as shown in FIG. 2, are provided with a step portion for holding an edge portion of the integrated composite electrode 2 and a rectangular opening at a central portion. The upper holder 3 is provided with a pair of fixtures 8 and 16 × 4 pairs of contact fittings 9. FIG. 5A is a top view of the holders 3 and 4 fixed with the integrated composite electrode 2 interposed therebetween, and FIG. 5 is a side view (BB sectional view) thereof.
FIG. 6B is a perspective view as viewed from the back side, and FIG. As can be seen from these figures, the fixture 8 is pivotally supported on two opposing sides of the upper holder 3 by shaft pins 8a. Grooves 4a are formed on two opposite sides of the lower surface of the lower holder 4, and the upper and lower holders 3, 4 sandwich the integrated composite electrode 2 by fitting the ridges 8b of the fixture 8 into the grooves 4a. It is firmly fixed in the state.

The 64 contact fittings 9 provided on the upper holder 3 so as to correspond to the electric contacts 7 of the integrated composite electrode 2 include:
It is formed by processing a good conductive metal plate having high elasticity, such as one obtained by plating Ni and Au on BeCu, and has a shape as shown in FIG. That is, it comprises a pin 9a, its base 9b, and a movable contact 9d extending from this base 9b via a curved portion 9c. With such a structure, the movable contact portion 9d can be elastically displaced with respect to the base 9b. The upper holder 3 has 64 (16 × 4) holes formed with holes through which the pin portions 9a of the contact fitting 9 are inserted and grooves into which the base portions 9b are fitted.

As shown in FIGS. 2 and 5B, the pin 9a protrudes 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 9b, 16 pins 9a protruding from the upper holder 3 are provided.
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 connection to the outside.

On the other hand, the movable contact portion 9d of the contact fitting 9 projects from the lower surface of the upper holder 3 in a state where the contact fitting 9 is inserted and fixed in the holes and grooves of the upper holder 3. This condition is better illustrated in FIG. 8, which is an assembly view from the opposite side to the assembly view of FIG. In a state where the holders 3 and 4 are fixed with the integrated composite electrode 2 interposed therebetween, the movable contact portion 9d of each contact fitting 9 comes into contact with the electric contact 7 of the integrated composite electrode 2 and is predetermined by the elastic deformation of the curved portion 9c. Is applied to the contact portion. Thus, the integrated composite electrode 2
The electrical contact 7 connected to the microelectrode 11 via the conductive pattern 12 has a small contact resistance (30
(Below the millimeter).

Next, the printed wiring board 5 will be described. The printed wiring board 5 fixes the assembly of the integrated composite electrode 2 and the holders 3 and 4, and extends from the microelectrodes 11 of the integrated composite electrode 2 to the conductive patterns 12, the electrical contacts 7, and the contact fittings 9. It also serves to bring out the electrical connection to the outside through the connector. Further, it also has a function of facilitating handling of a set or the like on a measuring device.

The printed wiring board 5 is formed using a glass epoxy substrate having a double-sided pattern. On the back surface shown in FIG. 8, connectors 5a are provided at four places around a circular opening formed at the center. ing. Upper holder 3
By inserting 16 pins 9a projecting in two rows in a staggered manner from four locations on the surface of the printed wiring board 5, the assembly of the integrated composite electrode 2 and the holders 3, 4 is inserted into the corresponding connector 5a. And are electrically connected.

On both side edge portions 5b of the printed wiring board 5, 2.54 mm pitch electrical contacts for double-sided edge connectors are formed, and these electrical contacts and the central connector 5a are formed.
And are connected by a lead pattern 5c. The inner row of the two-sided connector 5a is drawn out with a front surface pattern, and the outer row is drawn out with a back pattern, and 32 electrical contacts are formed on each of the edge portions 5b on both front and rear faces, thus a total of 64 electrical contacts. To secure the mechanical fixation, the upper holder 3 is fixed to the printed wiring board 5 by screws.
Can also be fixed.

FIG. 9 shows a preferred embodiment of the cell potential measuring device constituted by using the integrated cell setting device 1 constituted as described above. The measuring device of the present embodiment includes an integrated cell setting device 1 described above, an optical observation unit 20 including an inverted microscope 21 for optically observing cells set in the integrated cell setting device 1, A computer 30 includes 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 is provided with an inverted microscope 21 (IMT manufactured by Olympus) on which the integrated cell setting device 1 is set.
-2-F or equivalent to IX70), SI for microscope
T camera (C2400-08 or equivalent, manufactured by Hamamatsu Photonics)
22, a high-definition display 23, and an image file device (Matsushita Electric TQ-2600 or FTQ-3100F
24) is included. However, the high-definition display may double as the display of the computer 30. It should be noted that the specific devices shown in the parentheses above are examples, and the present invention is not limited to these. The same applies hereinafter.

As the computer 30, a personal computer compatible with WINDOWS equipped with an A / D conversion board and measurement software is used. A / D
The conversion board includes the A / D converter 31 and the D / A converter 3 shown in FIG.
Contains 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 means for applying a stimulus signal to cells and means for processing signals detected from cells, but also optical observation means (SIT camera and image file device) and It also controls the cell culture means. less than,
The main specifications of the measurement software are explained for each screen.

On the parameter setting screen, complicated stimulus conditions can be set by drawing a stimulus waveform on the screen using a keyboard or a mouse. The recording conditions are set by setting the number of input channels to 64 and the sampling rate to 10.
It is possible to cope with continuous recording for several hours at kHz. Further, the designation of an electrode to which a stimulus signal is applied or an electrode for extracting a detection signal from a cell can be designated by pointing a microscope image displayed on the screen with a mouse or a pen. In addition, setting of various conditions such as temperature and pH of the cell culture means 40 can be performed using a keyboard.

On the recording screen, spontaneous action potentials or induced potentials detected from cells are displayed in real time at a maximum of 64 channels. In addition, the recorded spontaneous action potential or evoked potential can be displayed so as to be superimposed on a microscopic image of the cell. In the case of evoked potential measurement, the entire recorded waveform is displayed. In the case of spontaneous action potential measurement, a recorded waveform is displayed only when spontaneous activity has been detected by a spike detection function using a window discriminator or a waveform discriminator. Along with displaying the recording waveform, measurement parameters (stimulation conditions, recording conditions, temperature, pH, etc.) during recording are also displayed in real time. An alarm function is also provided when the temperature or pH goes 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, a current source density analysis function, and the like. These analysis results can be displayed so as to be superimposed on the microscope image stored in the image file device.

When a 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, a stimulus signal is applied between two selected points among 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
(Equivalent to B-610J) 34 and the A / D converter 31 to be 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 an induced potential by a stimulus signal, the frequency band is set to 100 Hz to 10 kHz by a low cut filter.

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

Although the preferred embodiment of the cell potential measuring device according to the present invention has been described above, the cell potential measuring device according to the present invention is not limited to the above-described embodiment. Implementation is possible.

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

Further, since the cell culturing means 40 is provided in the measuring device, continuous measurement can be performed for a long period of time. However, the sample cells can be integrated in an incubator separately provided from the measuring device. The integrated cell setting device may be taken out of the incubator and set in the measuring device only during a relatively short measurement. In this case, the cell culture means 40 does not need to be provided in the measuring device.

Using the cell potential measuring device as described above,
An example in which a potential change associated with the activity of a nerve cell or a tissue actually cultured on an integrated cell placement device will be described below. Using a slice of rat cerebral cortex as nerve tissue,
The cells were cultured by the method shown in the culture method examples described below.

First, the result of comparison between the voltage waveform measured using the integrated cell setting device of the present apparatus and the voltage waveform measured using the conventional general-purpose glass electrode (electrode for measuring extracellular potential) will be described. Keep it. Measurement was performed 14 days after the start of culture.
The aged nerve tissue 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 temporal change of the evoked potential induced by eight nearby electrodes was measured. For comparison, the same voltage waveform was measured by sequentially moving the glass electrodes to the vicinity of the eight electrodes using a three-dimensional micromanipulator.

As described above, the voltage waveform measured using the integrated composite electrode (integrated cell setting device) at eight locations and the waveform measured using the glass electrode were compared. It was well approximated in some places.
Typical examples are shown in FIGS. 10A and 10B. FIG.
(A) shows a waveform measured by the integrated composite electrode, and FIG.
(B) shows a waveform measured by a glass electrode. Comparing the two waveforms shows that there is a slight difference in frequency characteristics. The measurement using a glass electrode has a slightly impaired ability to follow a rapid change in potential as compared to the measurement using an integrated composite electrode (planar electrode). This difference is considered to be due to a difference in electric capacity between the glass electrode and the planar electrode.

Next, a description will be given of the results of an experiment for examining the relationship between the number of days of culture of nerve cells cultured on the integrated cell placement device and the distribution of potential due to cell activity. Prior to culturing the cells, the surface of the integrated composite electrode was covered with collagen gel in order to enhance the adhesion between each electrode of the integrated composite electrode and the cell. That is, a collagen gel having a thickness of 50 μm or less was formed on the surface of each electrode coated with platinum black as described above and on the surface of the insulating coating therearound. Then, a slice (500 μm or less in thickness) of the rat cerebral cortex was placed on the collagen gel and on the microelectrode and cultured. FIG. 11 shows the measurement results of the self-power generation position, and FIG. 12 shows the measurement results of the evoked potential when a stimulus signal is given.

FIG. 11 (A) shows a microscope image of the sample cell and the microelectrode. In this image, the waveforms of the self-generated power measured at the seven electrodes indicated by 1 to 7 are shown. 11 (B) and (C). FIG. 11 (B)
FIG. 11C shows the waveform on the 6th day after the culture, and FIG.
It is a waveform on the 0th day. The scale of the microscope image, the time of the measured waveform, and the scale of the voltage 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 there is almost no synchronization between the electrodes. It can be seen that the electrodes become active at the same time and the synchronism between the electrodes is increased.

FIG. 12A also shows a microscope image of the sample cells and the microelectrodes. Then, by the image processing included in the measurement software of the computer described above, the outline of the cell and the position of each electrode were drawn on the screen from this microscope image, and the voltage waveform measured at each electrode was displayed in an overlapping manner. Things are shown in FIGS. 12B and 12C. FIG. 12 (B) shows the distribution of evoked potentials on day 5 after culture, and FIG. 12 (C) shows the distribution of evoked potentials on day 10 after culture. A pair of electrodes indicated by + and-on the upper right are the electrodes 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 large vertical swing at the left end is an artifact that directly corresponds to the stimulus signal,
A subsequent change in potential is indicative of actual cell activity.
From this measurement result, for example, on the 5th day after the culture, the cell activity is relatively limited to the vicinity of the electrode position to which the stimulus signal was applied, but on the 10th day after the culture, the cell activity was observed over a wide range. It can be seen that the size (amplitude) increases. [Examples of cerebral cortex culture method] (1) Medium 1: 1 of Dulbecco's modified Eagle medium and Ham F12 medium
The following additives were used in a mixed medium (GIBCO Co., LTD. 430-2500EB). * Glucose (glucose, GIBCO 820-5023IN) 2.85mg / L (Total of 6mg / L when combined with the one originally contained in the above medium) * Putrescine (putrescine, SIGMA Co., LTD. P5780) 10
0μM * Progesterone (progesterone, SIGMA P8783) 20nM * Hydrocortisone (hydrocortisone, SIGMA H0888)
20nM * Sodium selenite (WAKO Co., L
TD. 198-0319) 20 nM * Insulin (insulin, SIGMA 16634) 5 mg / L * Transferrin (transferrin, SIGMA T1147) 100 mg /
L * Sodium bicarbonate 2.438mg / L * Add appropriate amount of 1N HCl or 1N NaOH to adjust to pH 7.4.

After adding the above additives and sterilizing by filtration,
And stored for use. Hereinafter, this medium is simply referred to as “medium”
Call. (2) Configuration of well on integrated composite electrode Culture of nerve cells or nerve tissue on integrated composite electrode
Police with 22mm inside diameter, 26mm outside diameter and 8mm height for convenience
A cylinder made of styrene was bonded by the method described below. (A) Polystyrene cylinder (22 mm inside diameter, 26 mm outside diameter, height
8mm) on the underside of one-part silicone adhesive (Dowconi
Coating 891 or Shin-Etsu Chemical KE-42RTV)
You. (B) Polystyrene center and glass substrate center of integrated composite electrode
Glue them together, making sure that the centers of the cylinders match
I do. (C) Leaving for 24 hours in an environment where dust does not easily enter
To harden the adhesive. (D) After immersion in 70% ethanol for 5 minutes, in a clean bench
Sterilize by air drying with
Prepare. (3) Electrode surface treatment In order to enhance cell adhesion on the surface of the integrated composite electrode, the following
A collagen gel was formed on the electrode surface by the method. The following operations
All cropping was performed under a sterile atmosphere. (A) Prepare the following solutions of A, B, and C and keep them on ice. A. 0.3vol.% Diluted collagen hydrochloride solution (pH3.0, Nitta Zerachi
Cellmatrix TypeI-A) Double Becco Modified Eagle Solution and Ham F12 Medium
Sodium bicarbonate in 1: 1 mixed medium (GIBCO 430-2500EB)
Without adding, make a 10-fold concentration of the liquid used normally,
Filtered and sterilized C.I. Bicarbonate for 100 mL of 0.05N sodium hydroxide solution
Dissolves 2.2 g of sodium and 4.77 g of HEPES (GIBCO 845-1344IM)
(2) Liquids A, B, and C were mixed at a ratio of 8: 1: 1 while cooling.
Mix. At this time, C is added after A and B are mixed well.
And mix further. (C) Integrated composite pre-cooled to about 4 ° C
Dispense 1 mL of the mixed solution of b) into the electrode wells,
After covering the pole surface evenly,
Remove the mixed solution as much as possible with a pet. By this operation
And a coating of a mixing liquid having a thickness of 50 μm or less is formed on the electrode surface.
Be composed. (D) The integrated composite electrode having the liquid film for mixing was heated at 37 ° C.
The mixture is allowed to gel by warming for 30 minutes with
Make up the collagen matrix. (E) Add 1 mL of sterilized water into the well of the integrated electrode.
After washing for about 5 minutes, remove
U. (F) The operation of (e) is repeated two more times (three times in total). (G) Dulbecco's modified a
1: 1 mixed medium (GIBC and ham FH12 medium)
O 430-2500EB) plus the above additives (but a
1mL dispensed excluding insulin and transferrin)
Temperature 37 ° C, relative humidity 97% or more, CO_TwoConcentration 5
%, CO maintained at 95% air concentration_TwoIn the incubator
Store for use. (4) Culture of nerve cells or nerve tissue Culture forms are roughly divided into two types. That is, God
Dispersion culture of transcellular cells and organ culture of neural tissue. Less than,
Each will be described. (4-1) Dispersion culture method of rat cortical visual cortex neurons The following operations were all performed in a bacterial atmosphere. (A) Fetus of SD rat 16-18 days after pregnancy
Of the brain was removed and ice-cooled Hanks balanced salt solution (GIBCO 450-1
250EB). (B) Cut visual cortex from brain in ice-cold Hanks balanced salt solution
And transfer to the minimum required medium (GIBCO 410-1100EB).
You. (C) Visual cortex can be formed in Eagle's minimum essential medium
And cut it into 0.2mm square at most
You. (D) Put the finely cut visual cortex into a centrifuge test tube.
Hanks with no calcium and magnesium
After washing three times with a balanced salt solution, the mixture is dispersed in an appropriate amount of the same solution. (E) 0.25% in the centrifuge test tube of (d) above
Calcium and magnesium dissolved trypsin
Add Hank's balanced salt solution without the solution and double the total volume. Loose
Keep the temperature at 37 ° C for 15 minutes with gentle stirring.
Then, an enzymatic reaction is performed. (F) The medium shown in the above (1-1) (including additives.
Below, abbreviated as “medium”), and further 10 vol.
Add the purified solution to the centrifuge tube that passed through the above e).
In addition, double the total amount. Burner tip
Use a glass pasteur pipette with a small aperture
Repeat pipetting quickly (up to 20 times)
Isolate individual cells. (G) 9806.65m / sec^Two(Ie 1000
g) for 5 minutes. After completion of centrifugation, supernatant
Discard and suspend the precipitate in medium containing 5% fetal calf serum
You. (H) Repeat the above (g) two more times (three times in total). (I) The final obtained precipitate was subjected to 5 vol.% Fetal bovine serum.
Suspension in a medium containing red blood cells
Measure using a plate. After the measurement, the cells are
Concentration is 2-4 × 10⁶Adjust so that the number of cells / ml is reached. (G) CO after the treatment of the above (1-3)_TwoIncube
Take out the integrated composite electrode stored in the
Medium in the well (but insulin and transfer
(No phosphorus) and remove 5% of the calf fetus
Dispense 500 μL of medium containing serum. In addition,
Gently add 100 μL of the cell suspension after cell concentration adjustment,
Again CO_TwoLeave in the incubator. (R) Three days after the operation of (NU), half of the medium was renewed.
Replace with a new one. Replacement medium contains fetal calf serum
No medium was used. To lower the concentration of fetal bovine serum
Therefore, the proliferation of cells other than nerve cells (eg, clear cells)
Reduce breeding. (Ii) Thereafter, the same medium exchange is performed 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 second day after birth, and iced.
Hanks equilibrium with cold 0.25vol.% D-glucose
Soak in salt solution. (B) Ice-cooled HB containing 0.25 vol.% D-glucose
In SS, cerebral cortex injured brain membrane attached to brain
Be careful not to use sharp tweezers
Remove. (C) 500 μl from the corpus callosum on one side of the cerebral cortex excluding the brain membrane
about m, using small scissors for ophthalmic surgery,
Cut from the occipital lobe to the frontal lobe along the corpus callosum
You. (D) Then, using micro scissors for ophthalmic surgery,
(C) Large with a thickness of 200 to 300 μm perpendicular to the cut surface
Cut the brain cortex and make sections. (E) Furthermore, using micro scissors for ophthalmic surgery,
Is adjusted to about 1 × 1. (F) Prepared in “(3) Electrode surface treatment” above
Integrated composite electrode with CO _TwoRemove from incubator
Then, adjust the size of the cerebral cortex section to a diameter of 2 mm or more.
Gently suck up the pipette without damaging it.
Transfer into the culture well of the combined electrode. (G) Pasteur with burner smoothed tip
Be careful not to damage the cerebral cortex section using 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 slice on the integrated composite electrode,
Adjust the volume of the ground, the bottom of the section touches the culture medium,
To touch. (I) After adjusting the amount of medium, put the integrated composite electrode in a sterile Petri dish.
And 5 m of sterile water at 37 ° C to prevent drying of the medium.
1 into a Petri dish,_TwoIn the incubator
Let stand still. (Nu) After that, pay attention to the amount of medium and replace the medium once a day.
went. The amount of medium is the same as in (h) above.
You.

[0046]

As described above, according to the cell potential measuring device of the present invention, the living cells set in the integrated cell setting device can be simultaneously activated at a plurality of locations without damaging the living cells. The potential can be measured. And
Not only can the self-generated potential be measured from any of a plurality of electrodes, but also the measurement of the evoked potential obtained by applying a stimulus between any of the electrodes can be simultaneously performed with the plurality of electrodes.
In addition, by processing using a computer or the like, for example, from the microscope image obtained by the optical observation means, while confirming the relationship between the sample cell and the electrode position, it is possible to determine electrodes to which a stimulus signal is applied, It is also possible to superimpose on the image showing the cell and electrode position,
This can greatly contribute to making experiments in the field of electroneurophysiology efficient and accurate.

Further, by providing a cell culture means for maintaining the culture atmosphere of the cells set in the integrated cell setting device, continuous measurement over a long period can be performed in a stable state with the sample cells set in the measuring device. be able to.

[Brief description of the drawings]

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

Fig. 2 Assembly drawing of integrated cell installation device

FIG. 3 is a plan view showing 64 microelectrodes and a drawing pattern provided at the center of an integrated composite electrode constituting 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 cross-sectional view showing a state in which the integrated composite electrode is fixed between the upper and lower holders.

FIG. 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 the 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. 2;

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

FIG. 10 shows a voltage waveform due to the activity of cultured cells measured using the integrated cell setting device used in the apparatus of the present invention, and a voltage measured using a conventional general-purpose glass electrode (electrode for measuring extracellular potential). Diagram showing an example of comparison with a voltage waveform

FIG. 11 is a view showing a measurement result of a self-power generation position of a cultured cell measured using the apparatus of the present invention.

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

[Explanation of symbols]

 DESCRIPTION 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

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 33/483 G01N 27/46 341M (72) Inventor Makoto Takeya 1006 Ojidoma, Kadoma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Invention 1006 Kazuma Kadoma, Kazuma, Osaka Pref. Matsushita Electric Industrial Co., Ltd. (56) References JP-A-6-78889 (JP, A) JP-A-3-246457 (JP, A) JP-A Sho51 -94893 (JP, A) JP-A-7-59554 (JP, A) Japanese Utility Model Application Laid-Open No. 50-51668 (JP, U) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/48 A61B 5/04 G01N 1/28 G01N 27/28 341 G01N 27/416 G01N 33/483

Claims (13)

(57) [Claims] 1. A cell potential measuring device for measuring the electrophysiological properties of cells, comprising: A plurality of microelectrodes are provided on a substrate, and a cell mounting portion for mounting cells on the microelectrodes is further provided .
Connected to the conductive pattern and the end of the conductive pattern
B. an integrated composite electrode with a defined electrical contact; Electrical connection hands stage for deriving an electrical signal from the integrated multiple electrode while applying an electrical signal to the integrated multiple electrode, C. A stimulus signal application means connected to said electric connection means B for applying an electric stimulus to said cells; B. a signal processing means connected to said electrical connection means for processing an output signal from the electrophysiological activity of said cells ; The electrical connection means is a contact that contacts the electrical contact.
Hold the glass plate from above and below and fix it
A cell potential measuring device comprising a two-piece holder . 2. The cell potential measuring device according to claim 1, further comprising an optical observation means for optically observing said cells. 3. A cellular potential measuring apparatus according to claim 1 or 2, further comprising a cell culturing means for maintaining a culture atmosphere of the cells. 4. The cell potential according to claim 3, wherein the cell culturing means comprises a temperature adjusting means for maintaining a constant temperature, a means for circulating a culture solution, and a means for supplying a gaseous mixture of air and carbon dioxide. measuring device. 5. The cell potential measuring apparatus according to claim 1, wherein 64 electrodes are arranged in 8 columns and 8 rows as said plurality of microelectrodes. 6. An electrode area of each of said microelectrodes is 4 ×
The cell potential measurement device according to claim 1, wherein the cell potential measurement device is within a range of 10 ² to 4 × 10 ⁴ μm ² . 7. The printed wiring board according to claim 1 , wherein the electric connection means further includes 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. Cell potential measurement device. 8. The cell potential measuring device according to claim 7 , wherein the contact resistance between the electrical contact and the contact fitting and the contact resistance between the contact fitting and the connector are each 30 milliohms or less. 9. The cell potential measuring device according to claim 2 , wherein said optical observation means includes an optical microscope, and an imaging device and an image display device connected thereto. 10. The cell potential measuring device according to claim 9 , wherein said optical observation means further comprises an image storage device. 11. The cell potential measuring device according to claim 1, wherein said stimulus signal applying means is a pulse signal generator. 12. The multi-channel amplifier according to claim 1, wherein said signal processing means includes a multi-channel amplifier for amplifying an output signal due to electrophysiological activity of said cells, and a multi-channel display device for displaying the amplified signal waveform in real time. The cell potential measurement device according to claim 1. 13. The method according to claim 1, further comprising: outputting the stimulus signal via a D / A converter; inputting and processing an output signal based on the electrophysiological activity of the cell via an A / D converter; 4. The cell potential measuring device according to claim 3 , further comprising a computer for controlling the cell culture means.