JP2002522028A - Assembly and device for extracellular electrophysiological recording and use thereof - Google Patents

Abstract

(57) Abstract: The present invention relates to an assembly comprising a field effect transistor having vertebrate cells, particularly mammalian cells, more specifically, a cardiomyocyte immobilized thereon, and extracellular electrophysiology comprising the assembly. Recording devices and their pharmaceutically useful compounds, such as compounds that may have cardiophysiological efficacy, or compounds that may have side effects, or may be toxic Use for testing potential compounds.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

【Technical field】

The present invention relates to an assembly comprising a field effect transistor having vertebrate cells, in particular mammalian cells, more particularly cardiomyocytes immobilized thereon, an extracellular electrophysiological recording device comprising the assembly, and Those compounds that are pharmaceutically useful, such as compounds that may have cardiophysiological efficacy, or that may have side effects, or that may be toxic. For use to test.

[0002]

[Background Art]

The propagation of impulses in myocardial tissue depends on the active properties of cardiomyocytes (including ion channels, ion exchangers, and ion pumps) and the passive properties (membrane resistance and membrane capacity, individual cell size and shape, cell aggregation, gap junctions) And the spatial composition of the extracellular space). The appearance of spontaneous and rhythmic electrical and mechanical activities in cultured cardiomyocytes is widely used in studies of cardiac physiology. Electrical activity in tissue culture is usually achieved by inserting a glass micropipette into the cells,
It is recorded by a patch clamp pipette or a metal microelectrode. However, using these techniques for an extended period of time is difficult because they are displaced, damaged, or damaged. Twenty years ago, efforts have begun to stimulate and record without invading cultured cells using microcircuits embedded in substrates. Using semiconductor planar technology, metal microelectrodes (MMEs) embedded in substrates of various sizes and shapes have been created for long-term recording from cardiac cells and neurons. Field effect transistors (FETs) have also been used to record from electrically active cells and tissues. A large FET with an unmetallized gate is used to record extracellular voltage from muscle tissue,
FET arrays with "floating" gold gates are used to record from hippocampal slices. More recently, individual invertebrate neurons have been recorded using open-gate FETs.

[0003]

DISCLOSURE OF THE INVENTION

As mentioned above, the present invention records the electrical activity in each of the cultured vertebrate cells, thereby enabling, for example, studying cardiac physiology.
It is an object to provide a device based on an assembly based on a FET or an assembly based on such a FET, wherein such an assembly or a device including an assembly based on such a FET is Should not be affected by cell movement, breakage, or damage for long periods of time.

[0004] The present invention further relates to FET-based assemblies, or such FETs, each of which may be used to test compounds that may be pharmaceutically effective.
It is an object of the present invention to provide a device consisting of an assembly based on the above.

[0005] The above technical problem is solved by providing the embodiments characterized in the claims. Other features and advantages of the invention will be apparent from the description and drawings of the preferred embodiments.

In particular, according to the present invention, at least one field effect transistor consisting of a source contact and a drain contact, and an unmetallized surface region of the field effect transistor between the source contact and the drain contact. Provided is an assembly comprising one or more layers of contacting vertebrate cells, particularly mammalian cells, and more particularly cardiomyocytes.

The unmetallized surface area covers at least part of the channel of the field effect transistor, the conductivity of which can be influenced via a field effect by a layer of vertebrate cells. Therefore, the non-metallized surface region coincides with at least a part of the non-metallized gate region of the field effect transistor.

[0008] In one embodiment, a monolayer of cardiomyocytes is immobilized on the unmetallized surface area of the field effect transistor. Preferably, the fixed cells are present in a serum-containing culture. In one preferred embodiment, the cardiomyocytes are beating rat cardiomyocytes, and the cell density is, for example,
It is in the range of 10 ⁴ -10 ⁶ pieces / cm ² .

[0009] In another preferred embodiment of the present invention, the field effect transistor comprises n
Alternatively, it is a p-channel silicon field effect transistor. In this case, the surface region that has not been metallized corresponds to at least a part of the surface region of the oxidized gate of the silicon field effect transistor.

[0010] In another preferred embodiment of the present invention, the field effect transistor includes at least one III-V or II-V semiconductor heterostructure. For example, a III-V semiconductor high electron mobility transistor (HEMT) may include a non-metallized surface area formed on the semiconductor surface between the source and drain contacts or on any protective oxide surface. Can be used with.

The size of the gate region of the field-effect transistor that has not been metallized is preferably in the range of 28 × 9 μm ² to 10 × 1 μm ² .

[0012] In another preferred embodiment of the present invention, the field-effect transistor comprising a surface region that has not been metallized is a matrix in which each center is separated by a fixed distance, preferably each center is 200 μm. They are arranged in a 4 × 4 matrix separated from each other.

In another embodiment of the invention, a means for amplifying an electrical signal detected at the aforementioned assembly and optionally at a recording terminal such as a source contact or a drain contact of the assembly and / or Means for monitoring the electrical signal, such as a computer connected to the device, for providing an extracellular electrophysiological recording device. Such a device with the additional means of measuring the time delay between said electrical signals is advantageously used to characterize the burst pattern of the signal in a layer of vertebrate cells, for example cardiomyocytes can do.

[0014] Like the device described above, the above-mentioned assembly may also be a compound that may be pharmaceutically active, for example a compound that may have cardiophysiological efficacy, or a compound that may have side effects. It can be conveniently used to test objects or potentially toxic compounds. Of course, compounds consisting of at least two of the different types of compounds can also be tested by the assembly, as well as the devices described above.

[0015] For example, the assembly or the device according to the present invention, respectively, pulsates the heartbeat by contacting such a potential compound to see if the potential compound causes an arrhythmia. For online monitoring of which compounds can restore the heartbeat after artificially arrhythmicizing heart cells, for example by adding calcium, It can be used as a “biochip sensor”.

Further, when each of the assembly or the device according to the present invention is used,
The source, direction and speed of the burst pattern between cells can be determined for the diagnosis of a potential heart disease patient.

The following examples illustrate the invention in more detail, but do not limit the invention.

[0018]

【Example】

Experimental Equipment The field effect transistor (FET) arrays used in this example were fabricated using standard silicon planar technology. The array is comprised of 16 p-channel FETs having a non-metallized gate region that matches a non-metallized surface region. The size of the gate or gate region is in the range of 28 × 9 μm ² to 10 × 1 μm ² , and each center is arranged in a 4 × 4 matrix separated by 200 μm. These chips use standard 28DIL ceramic chip carriers (NGK Spark Plug Co., LT
D, Japan), wire bonded, silicone polymer (Sylga
rd 184 and 96-083, Dow Corning).

The capsule-shaped device, together with the glass ring fixed on the chip carrier, forms a small culture dish. The electric measurement using the FET was performed using an Ag / AgCl wire as a reference electrode for specifying a gate potential. drain·
Source-to-source voltage (V _DS ) is -2.0 V, and Ag / AgCl-source voltage (V _GS ) is -2.0 V
Under normal driving conditions, the transconductance (dI _DS / dV _GS ) was between 0.1 ms and 0.4 ms depending on the gate size. Under these conditions, the leakage current from the source ( _IGS ) was negligible.

Heart elastic cells were prepared according to the procedure described in detail in Bhatti et al., J. Mol. Cell. Cardiol. 1989, 10, 995. Briefly, hearts were removed from 1 to 3 day old rats, minced, separated, and thinly placed on a recording device in culture containing serum. The FET array is washed with 25% sulfuric acid before being covered,
Washed with li-Q water, sterilized with 70% ethanol and covered with fibronectin for about 1 hour. Finally, after washing with sterile water, approximately 80 μl of 5 × 10 ⁵ to 9 × 10 ⁶
Fill the culture dish of the FET array with a suspension containing cells / ml cells,
The cell density was approximately 3 × 10 ⁷ cells / cm ² . The culture was cultured at 37 degrees in a humidified atmosphere. Under these conditions, within a few days, a confluent monolayer of cells exhibiting spontaneous rhythmic activity grew. The electrical activity of these cells was examined from day 3 after the thin coating.

For recording, the FET array was connected to a 16-channel preamplifier mounted under a microscope. Offset currents resulting from the drive states of all 16 channels were compensated and the recorded signal was amplified with a gain of 100. Up to four selected channels were monitored using a computer connected to the electronics. In some cases, extracellular and intracellular recordings were made simultaneously using glass microelectrodes filled with 3M KCl and mounted on the head stage of a holder preamplifier (Luigs & Neumann, Germany). For intracellular recordings, the headstage signal is a whole cell amplifier (Lis) that can be connected to the computer and oscilloscope.
t electronic, Germany). The temperature was kept constant at 37 degrees using a heater pad attached to the FET preamplifier. FIG. 1 schematically shows a main part of the experimental apparatus described above.

Results and Data Analysis A. Single Point Recording In fully grown cultured heart cells, action potentials are generated by currents of sodium, potassium, calcium and chloride compounds passing through the cell membrane. Inward sodium currents are the first trigger for a rapidly rising action potential. The calcium current results in a flat phase of the action potential, and the potassium current repolarizes the cell.

FIG. 2 shows a typical recording from cardiomyocytes taken simultaneously using intracellular microelectrodes (upper pattern) and FETs (lower pattern). Intracellular recordings were taken from cells grown several μm away from the FET recording point. A more detailed chart is shown in FIG. A rapid rise in the intracellular voltage V _M (microelectrodes, FIG. 3A) initially causes a spike in the source-drain current I _DS , followed by a long-lasting decrease (FET, FIG. 3B).

A corresponding gate-source voltage V _J can be calculated from the transfer characteristics of each transistor, and an extracellular action potential having an amplitude of 0.2 mV to 0.5 mV is recorded. These voltages were 5 to 10 times greater than the background noise. The first spike in extracellular signal represents about 50% of the total signal. First 5 of long lasting second part of signal
From 0 ms to 100 ms seems to resemble the Na ⁺ spike and K ⁺ components of the action potential overshoot. The total duration of the second part of the extracellular signal was about 100 ms to 200 ms.

The extracellular voltage V _J (t) at the gate can be simulated by convolution of the intracellular voltage V _M (t) by the Fourier transform of the transfer function h (ω). FIG. 4 shows an equivalent circuit configuration for determining the transfer function. The first rapid rise of the extracellular signal is determined by convolving the intracellular voltage V _M (t) using a high-band filter element consisting mainly of the membrane capacitance C _M and the junction seal resistance R _J. You. Therefore, the shape and amplitude of this peak is mainly determined by the time constant (T _H = C _W · R _J ) of the high band filter element (FIG. 3C). The shape and magnitude of the extracellular signal should be described by a more complex point-contact or plate-contact model built to connect single invertebrate neurons with FETs using only passive membrane functional elements Is known to be impossible (D in FIGS. 3 and 6). In either case, in addition to the high-band filter element, a typical value of the membrane resistance R _M of the coupling portion and the capacitance C _G of the gate oxide is used.

The limitations of this model with respect to the second part are determined by the high-pass filter element, which means that this model cannot properly represent the transfer function. This part most likely to be due to the contribution of the current I _C which flows through the membrane in the contact area for active ion channels (Fig. 4). The current through the ion channel is determined by a voltage controlled channel with a time dependent resistance R _Chan = g _Chan .
The current I _C (t) flowing into the contact area is a time-dependent intracellular voltage V _M (t) (I _C (t) = R _Chan · V _M generated by the fixed resistance and the flow of ions through the membrane. It can be calculated by using (t)).

Applying Kirchhoff's law gives the following equation:

( V _M (ω) -V _J (ω)) (1 / R _M + ωC _M -1 / R _Chan ) = V _J (ω) (1 / R _J + ωC _G ) (1) h ₀ Using = R _J / R _M and K ₀ = R _M / R _Chan provides the following transfer function for the equivalent circuit shown in FIG.

H (ω) = (1 + h ₀ ωτ _L / h ₀ (1-K ₀ ) + ωτ _H +1) ⁻¹ (2) Current flowing into the junction due to active ion channels in the contact area Flows in the opposite direction of the current based on the passive element in the model circuit. The best simulation for the measured extracellular signal is shown in FIG. 3E. Simulation following _{parameters, T H = C M · R} J = 5μs, T L = C G · R M = 1ms, h 0 = 0.001, k 0 = 2.6 is used for the. Except for the last 250 ms of the signal, the curves simulated and measured using this additional active membrane functional element are in very good agreement. This last 250 ms intracellular signal is based primarily on the release of Ca ²⁺ ions in the cytoplasm that do not contribute to the extracellular signal. FIG. 5 shows the coefficients used in the simulation shown in FIG. 3 and the phase differences h (ω) of the transfer functions of various spectra. With the introduction of the active film functional element, the transmissivity is slightly increased at a low frequency, and the phase is shifted by 180 degrees.

In addition to the above signals, the action potential of the elastic cells of the rat heart was recorded, but the peak of overshoot disappeared. Figure 6 shows intracellular microelectrode (A) and FET
(B) shows a typical form of these signals recorded simultaneously. In this case, the first part of the extracellular signal will be about 1 /
3 only contributed. The second part is more pronounced and lasts almost as long as the intracellular signal. The simulated curves are in good agreement with the measured data (FIG. 6E). The best simulations were obtained with the following parameters: T _H = C _M R _J = 10 μs, T _L = C _G R _M = 10 ms, h ₀ = 0.001, k ₀ = 2.5.

FIG. 7 shows the maximum signal recorded in this embodiment. The shape of the extracellular signal almost coincides with the intracellular signal (FIG. 6A). The amplitude of these signals, which lasted approximately 300 ms, was about 25 mV. The capacitive current is negligible, and the current flow is mainly determined by the ratio R _M / R _J. Such a signal can be explained by assuming that the seal resistance R _J of the coupling portion is within the range of the film resistance R _M in the coupling portion. This is the seal resistance
Or R _J is assumed to be the expected factor greater than 1000, it can be explained by the fact that the membrane resistance R _M is smaller than ordinary membrane resistance.

B. Multipoint Recording According to another embodiment of the present invention, simultaneous recording from various transistors is performed. FIG. 8 shows spontaneous extracellular activity from cardiomyocyte monolayers recorded by four different FETs. The location of the FET used for recording is shown in FIG. 8b. As shown in FIG. 3, all signals are similar in shape. The signal amplitude is different for each FET, probably due to varying coupling strengths. Due to the electrical connection between the cells in the layer, the cell with the highest repetition rate action potential determines the beating cycle of the entire layer. From the time delay between the recordings of action potentials at various points, the source, direction and speed of the burst pattern (approximately
0.2 m / s).

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a measuring device used for electrical recording from elastic cells of a rat heart.

FIG. 2 shows electrical recordings taken from rat heart elastic cells 4 days after culture.
The measurement was performed using FET (lower pattern) and intracellular microelectrode (upper pattern).
Sometimes has been done. Source-drain current I determined by the transfer characteristics of the FET _DS And effective gate voltage V_GIs shown in the lower pattern and is measured by the inserted microelectrode.
Fixed membrane voltage V_MAre shown in the upper pattern.

FIG. 3 is an enlarged view of the action potential of FIG. 2 recorded using a microelectrode (A) and an FET (B). The lower pattern shows a simulated curve using the equivalent circuit shown in FIG. 6, where (C) uses only the high-band filter element and (D) does not use the active membrane functional element. , And (E) show the case where an active film functional element is used, respectively.

FIG. 4A is a diagram showing an equivalent circuit configuration for explaining a measured signal. This model consists of the gate oxide capacitance C _G and the seal resistance R _J , the membrane capacitance C _M and the membrane resistance R _{M at} the junction, as well as the current contribution due to the active ion channel, b) the intracellular signal V _M which is a diagram showing an equivalent circuit configuration shown in the form of 4 electrodes which may be used to enable the gate-source voltage V _J by adding.

FIG. 5 is a diagram showing the phase difference h (ω) of a coefficient and a spectrum transfer function used to create a model of an extracellular signal recorded using a FET, and a curve A is a high-pass filter element. , Curve B includes passive membrane functional elements, and curve C includes active and passive membrane functional elements.

FIG. 6 is a diagram showing recordings taken simultaneously from elastic cells of the heart using intracellular microelectrodes (A) and FETs (B). The lower pattern shows curves simulated by applying the equivalent circuit of FIG. 4 (using a high-band filter element (c), no active membrane functional element (D), and with an active membrane functional element (E)).

FIG. 7 shows the maximum signal recorded from cardiomyocytes using FET.

FIG. 8 a) is a curve showing spontaneous extracellular activity from a cardiomyocyte monolayer recorded by four different FETs, and b) shows the position of the FET used for recording.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] October 11, 2000 (2000.10.11)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/483 G01N 33/50 Z 33/50 310Z (81) Designated countries EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK , EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT , UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference)

Claims (12)

[Claims] At least one field effect transistor including a source contact and a drain contact, and in contact with an unmetallized surface area of the field effect transistor between the source and drain contacts. An assembly comprising one or more layers of vertebrate cells. 2. The assembly according to claim 1, wherein said vertebrate cells are mammalian-derived cells. 3. The assembly according to claim 1, wherein said cells are cardiomyocytes. 4. The assembly according to claim 3, wherein said cardiomyocytes are beating rat cardiomyocytes. 5. The assembly according to claim 1, wherein the cell density is in a range of 10 ⁴ -10 ⁶ cells / cm ² . 6. The assembly according to claim 1, wherein said field effect transistor is an n or p channel silicon field effect transistor. 7. The method according to claim 1, wherein the field-effect transistor has at least one of III-V or I-V.
The assembly according to any one of claims 1 to 6, comprising an IV semiconductor heterostructure. 8. The semiconductor device according to claim 1, wherein the size of the non-metallized surface region of the field-effect transistor is in a range of 28 × 9 μm ² to 10 × 1 μm ^2. Assembly as described in. 9. The non-metallized surface area is arranged in a matrix in which each center is separated by a fixed distance, particularly in a 4 × 4 matrix in which each center is separated by 200 μm. 7. The assembly according to claim 6, wherein: 10. A means for amplifying said assembly according to any of claims 1 to 9 and optionally an electrical signal detected at a recording terminal of said assembly and / or means for monitoring said electrical signal. A device for extracellular electrophysiological recording, comprising: 11. The extracellular electrophysiological device of claim 10, further comprising means for measuring a time delay between said electrical signals to determine a signal burst pattern in a layer of vertebrate cells. Recording device. 12. The method according to claim 1, for testing a compound which may be pharmaceutically effective, has a side effect, or a compound which may be toxic. Use of the assembly according to any of the preceding claims, or the device according to claim 10 or 11.