ASSEMBLY AND APPARATUS FOR EXTRACELLULAR ELECTROPHYSIOLOGICAL RECORDINGS AND THEIR USE

Abstract: The present invention relates to an assembly comprising a field-effect transistor having vertebrate cells, particularly cells of mammalian origin, more particularly myocardial cells, fixed thereon, an apparatus for extracellular electrophysiological recordings comprising the assembly and its use for testing potential pharmaceutically effective compounds, such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or potentially toxic compounds.

Description: ASSEMBLY AND APPARATUS FOR EXTRACELLULAR ELECTROPHYSIOLOGICAL RECORDINGS AND THEIR USE Description The present invention relates to an assembly comprising a field-effect transistor having vertebrate cells, particularly cells of mammalian origin, more particularly myocardial cells, fixed thereon, an apparatus for extracellular electrophysiologi- cal recordings comprising the assembly and their use for testing potential pharmaceutically effective compounds, such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or potentially toxic compounds. Impulse propagation in the cardiac muscle tissue is determined by both active (including ion channels, ion exchangers, and ion pumps) and passive properties (membrane resistance and capacitance, size and shape of individual cells, cell assemblage, topology and density of gap junctions, and the spatial organization of the extracellular space) of the cardiac muscle cells. The presence of spontaneous rhythmic electrical and mechanical activity in cultured myocardial cells is widely used to study cardiac physiology. Electrical activity in tissue culture is usually recorded by intracellular glass micropipettes, patch clamp pipettes, or metal microelectrodes. However, it is difficult to use these techniques for extended periods because of movement, breakage, or cell damage. Efforts to noninvasively stimulate and record from cultured cells using substrate-embedded microcircuits began 20 years ago. By utilizing semiconductor planar technology substrate embedded metal microelectrodes (MME) of various size and shapes have been built for long term recording from cardiac cells and neurons. Field- effect transistors (FET) have also been used to record from electrically active cells and tissues. A large FET with a non-metallized gate was applied to record extracellular voltage from muscle tissue and FET arrays with "floating" gold gates have been used to record from hippocampal slices. More recently recordings form individual invertebrate neurons were made using FETs with an open gate. Thus, it is an object of the present invention to provide a FET based assembly or an apparatus comprising such a FET based assembly, respectively, which enables the recording of the electrical activity in cultured vertebrate cells, to thereby study, for example, cardiac physiology, wherein such an assembly or an apparatus comprising such a FET based assembly, respectively, should not be affected by cell movement, breakage, or damage for extended periods of time. A further object of the present invention is to provide a FET based assembly or an apparatus comprising such a FET based assembly, respectively, which can used for testing potential pharmaceutically effective compounds. The solution to the above technical problem is achieved by providing the embodiments characterized in the claims. Other features and advantages of the invention will be apparent from the description of the preferred embodiments and drawings. In particular, according to the present invention, there is provided an assembly, comprising at least one field-effect transistor comprising a source contact and a drain contact, and one or more layers of vertebrate cells, particularly cells of mammalian origin, more particularly myocardial cells, in contact with an non- metallized surface region of the field-effect transistor between the source con ¬ tact and the drain contact. The non-metallized surface region covers at least a portion of a channel of the field-effect transistor, the conductivity of which can be influenced via field-effect by the layer(s) of vertebrate cells. Hence, the non- metallized surface region corresponds to at least a portion of a non-metallized gate region of the field-effect transitor. In one embodiment, a monolayer of myocardial cells are fixed onto the non- metallized surface region of the field-effect transistor. The fixed cells are preferably present in serum-containing medium. In a preferred embodiment, the myocardial cells are beating rat cardiac muscle cells. The cell density ranges, for example, from 10 4 - 10 6 cells/cm 2 . In a further preferred embodiment of the present invention, the field-effect transistor is a n- or p-channel silicon field-effect transistor. The non-metallized surface region corresponds to least a part of a gate-oxide surface region of the silicon field-effect transistor in this case. In a further preferred embodiment of the present invention, the field-effect transistor comprises at least one lll-V or ll-V semiconductor heterostructure. For example a lll-V semiconductor high electron mobility transistors (HEMT) can be employed with the non-metallized surface region either being formed on the semiconductor surface between the source contact and drain contact or on an optional protective oxide surface. Preferably, the size of the non-metallized gate regions of the field effect transistors ranges from 28x9 μm 2 to 10x1 μm 2 . In a further preferred embodiment of the present invention, the field-effect transistors comprising the non-metallized surface regions are arranged in a matrix with spaced apart centers, preferably a 4x4 matrix with the centers 200 μm apart. In a further embodiment of the present invention, there is provided an apparatus for extracellular electrophysiological recordings comprising the aforementioned assembly, and optionally means for amplifying electrical signals detected at the recording terminals of the assembly, for example source and drain contacts, and/or means for monitoring the electrical signals, e.g. a computer connected to the electronic set-up. Such an apparatus with an additional means for measuring time-delays between the electrical signals can be advantageously used to determine the characteristics of a burst pattern of signals in the layer/layers of vertebrate cells, e.g. myocardial cells. The aforementioned assembly as well as the apparatus mentioned above can be advantageously used for testing potential pharmaceutically effective compounds, such as compounds having potentially cardiac physiologically effectiveness, or compounds having potential side effects, or pontentially toxic compounds. Of course, a combination comprising at least two of the aforementioned different types of compounds can also be tested by the assembly as well as the apparatus mentioned above. For example, the assembly or the apparatus, respectively, according to present invention can be used as an "biochip-sensor" for on-line monitoring whether potential compounds induce irregularities of pulse or whether the cardiac beat loses the rhythm by bringing in contact with such potential compounds or, otherwise, which compounds are capable of restoring the cardiac beat after the cardiac cells have been artificially set in arhythmic condition, for example by addition of calcium. Further, using the assembly or the apparatus, respectively, according to present invention, the origin, the direction and the velocity of burst pattern between the cells can be determined for the evaluation of potential cardiacs. The figures show: Fig. 1 is a schematic drawing showing the measurement setup used for the electrical recordings from rat cardiac myocytes. Fig. 2 shows the electrical recordings from rat cardiac myocytes after 4 days in culture, wherein the measurements have been performed simultaneously with FET (lower trace) and an intracellular microelectrode (upper trace). The source- drain current l DS , the effective gate voltage V G determined from the transfer characteristics of the FET are depicted in the lower trace and the membrane voltage V M measured by the impaled microelectrode is depicted in the upper trace. Fig. 3 shows the expansion of the action potentials in Fig . 2 recorded with an microelectrode (A) and an FET (B). The lower traces show the simulated curve using the equivalent circuit shown in Fig. 6: (C) using only the highpass filter elements, (D) without and (E) with active membrane elements. Fig . 4a shows the equivalent circuitry for the explanation of the measured signals. The model consists of the capacitance of the gate oxide C G in the junction and the seal resistance R Jt the capacitance C M and resistance R M of the membrane, as well as the contribution of the current due to active ion channels. Fig. 4b shows an equivalent circuitry presented in the four-pole configuration used for the determination of the effective gate-source voltage V by applying the intracellular signal V M . Fig. 5 shows the modulus and phase difference of the spectral transfer functions h{ω) used for the modelling of the extracellular signals recorded with FET: A) highpass filter elements, B) including passive membrane elements and C) including active and passive membrane elements. Fig . 6 shows the simultaneous recording from cardiac myocytes with an intracellular microelectrode (A) and an FET (B) . The lower traces show the simulated curves applying the equivalent circuit from Fig. 6 (highpass filter elements (C), without (D) and with (E) active membrane elements) . Fig. 7 shows the largest signals recorded from the heart muscle cells with an FET. Fig. 8a shows the spontaneous extracellular activity from a monolayer of cardiac muscle cells recorded kom 4 different FETs. Fig. 8b shows the position of the FETs which were used for the recordings. The following example illustrates in more detail the present invention, but should not be construed as limiting the invention. EXAMPLE Experimental setup Field-effect transistor (FET) arrays used in this example were fabricated using standard silicon planar technology. The array consisted of 1 6 p-channel FETs with non-metallized gate regions corresponding to non-metallized surface regions. The size of the gates or gate regions ranged between 28x9 μm 2 down to 10x1 μm 2 and were arranged in a 4x4 matrix with the centers 200 μm apart. These chips were mounted on standard 28 DIL ceramic chipcarriers (NGK Spark Plug Co., LTD, Japan), wire-bonded and encapsulated using a silicone polymer (Sylgard 1 84 and 96-083, Dow Corning) . Together with a glass ring fixed onto the chip carrier the encapsulated device forms a small culture dish. Electrical measurements with the FET were carried out with a Ag/AgCI wire as a reference electrode which defines the gate potential. At the usual driving conditions in which the drain-source voltage ( V DS ) was -2.0 V and the Ag/AgCI-source voltage ( V GS ) was -2.0 V the transconductance (d/ DS ld V GS ) was between 0.1 and 0.4 mS depending on the gate size. In these conditions the leakage current from the source (/ GS ) was negligible. Cardiac myocytes were prepared following a technique which has been described in detail in Bhatti et al., J. Mol. Cell. Cardiol. 1 989, 10, 995. Briefly the hearts were removed from 1 to 3 day old rats, finely minced and dissociated and plated onto the recording devices in serum containing medium. Prior to plating the FET arrays were cleaned with 25% sulfuric acid, washed with Milli-Q water, sterilized with 70 % ethanol and coated with fibronectin for about 1 h. Finally after washing with steril water approximately 80 μl of a suspension containing between 5x10 5 and 9x1 0 5 cells/ml were used to fill the culture dish of the FET array, which resulted in a cell density of approximately 3x10 7 cells/cm 2 . The culture was incubated at 37 °C in a humidified atmosphere. Under these conditions, within 2-3 days, a confluent monolayer of cells exhibiting spontaneous rhythmic activity developed. The electrical activity of these cells was studied starting on the 3rd day after plating. For recording, the FET array was connected to a 1 6-channel preamplifier mounted under a microscope. The offset currents arising from the driving conditions of all 1 6 channels were compensated and recorded signals were amplified by a gain of 1 00. Up to four selected channels were monitored using a computer connected to the electronic setup. In some cases simultaneous extracellular and intracellular recordings were made using glass micro electrodes filled with 3 M KCI and mounted in a holderpreamplifier headstage (Luigs & Neumann, Germany). In the case of intracellular recordings the headstage signal was amplified using a whole cell amplifier (List electronic, Germany) which could be connected to the computer and an oscilloscope. The temperature was kept constant at 37 °C using a heater pad fitted to the FET preamplifier. In Fig . 1 the principal setup for the described experiment is schematically shown. Results and Data analysis A. Single site recordings In fully developed cultured cardiac cells the action potential is generated by the sodium, potassium, calcium and chloride currents across the cell membrane. The inward sodium current is the initial event triggering a fast rising action potential. The calcium current carries the plateau phase of the action potential and the potassium current repolarises the cell. Fig. 2 shows typical recordings from cardiac muscle cells performed simultaneously with an intracellular microelectrode (upper trace) and an FET (lower trace) . Intracellular recordings were made from cells grown several μm away from the recording site of the FET. A more detailed view of the recordings is shown in Fig. 3. The fast rising of the intracellular voltage V M (microelectrode, Fig. 3a) causes first an sharp increase followed by a long lasting decrease in the source-drain current l os (FET, Fig. 3b). From the transfer characteristics of each transistor, the corresponding gate- source voltage V could be calculated, and extracellular action potentials with amplitudes of 0.2 mV to 0.5 mV have been recorded. These voltages were between 5 and 1 0 times greater than the background noise. The sharp rise of the extracellular signals at the beginning represents about 50 % of the overall signal. The first 50 - 100 ms of the long lasting second part of the signal seems to resemble the overshooting Na + -spike and K + component of the action potential. The overall duration of the second part of the extracellular signal was about 100 to 200 ms. The extracellular voltage V j (f) at the gate can be simulated by a convolution of the intracellular voltage V M (t) by means of the Fourier transform of the transfer function (ω) . The equivalent circuitry for determination of the transfer function is shown in Fig. 4. The first, fast rising part of the extracellular signal is mainly determined from the convolution of the intracellular voltage V M (t) with the high- pass filter element consisting of the capacitance of the membrane C M and the seal resistance R j of the junction. Therefore, the shape and amplitude of this peak is mainly determined by the time constant (τ H = C w ■ RJ of the high-pass filter element (Fig. 3C) . It has been found that the shape and size of the extracellular signals could not be explained by means of the more complex point- contact or the plate-contact-model developed for the coupling of single invertebrate neurons with FETs using only passive membrane elements (Fig . 3 and 6, D) . In both cases there has been used in addition to the highpass filter element typical values of the capacitance of the gate oxide C G and the resistance R M of the membrane in the junction. The limit of this model in regard of the second part is determined by the highpass filter element, which means that it can not represent the proper transfer function. It is assumed that this part comes mostly from the contribution of current l c flowing across the membrane in the contact area due to active ion channels (Fig. 4) . The current through the ion channels is determined by voltage-controlled channels with a time dependent resistance R Chan = g Chan .T e current l c (t) flowing into the contact area can be calculated by using a fixed resistance and the time dependent intracellular voltage V M (t) (l c (t) = Rcn an ' V M (t)) caused by ion flow through the membrane. Applying Kirchhoffs law leads to the following equation: (Y M (ω)-^(ω))( - + ''ωC M --^)=Y J (ω)(-l- + /ωC G ) Using results in the following transfer function for the equivalent circuit shown in Fig . 4: y Current flowing into the junction due to active ion channels in the contact area is directed against the current based on the passive elements in the model circuit. The best simulation for the measured extracellular signal is shown in Fig. 3E. For the simulation the following set of parameters has been used: r H = C M R j = 5 μs; τ L = C G R M = 1 ms; h 0 = 0.001 ; k 0 = 2.6. Except for the last 250 ms of the signal a very good agreement between the simulated and the measured curve with this additional active membrane element has been achieved. The intracellular signal of these last 250 ms is mainly based on Ca 2 + ion release inside the cytoplasma which will not contribute to the extracellular signal. In Fig . 5 the modulus and the phase difference of the various spectral transfer functions h(ω) is displayed used for the simulations shown in Fig. 3. The introduction of the active membrane elements leads to a slightly increased transfer ratio and a phase shift of 1 80° at lower frequencies. In addition to the signals described above, action potentials of the rat cardiac myocytes have been recorded, wherein the overshooting peak was missing. Fig. 6 shows the typical shape of these signals recorded simultaneously with an intracellular microelectrode (A) and an FET (B) . In this case the first part of the extracellular signal contributes only about 1 /3 of the total amplitude due to the slower rise time. The second part is more pronounced and lasts almost as long as the intracellular signal does. The simulated curve shows a very good agreement with the measured data (Fig. 6E) . The best simulation was obtained by using the following set of parameters: r H = C M R j = 10 μs; τ L = C G R M = 10 ms; h 0 = 0.001 ; k 0 = 2.5. Fig. 7 shows the largest signal recorded in this example. The shape of the extracellular signal is almost identical to intracellular signals (Fig. 6A) . The amplitude of these signals which lasted approximately 300 ms, was about 25 mV. The capacitive current is negligible and current flow is determined mainly through the ratio R R j . This kind of signal can be explained by assuming that the seal resistance R d of the junction is in the range of the membrane resistance R M in the junction. This can be explained either by assuming that the seal resistance R d is a factor 1 000 larger than expected or by the fact that the mem- brane resistance R M is small compared to the usual membrane resistance. B. Multi Site Recordings According to another embodiment of the present invention, simultaneously recordings from various transistors have been carried out. Fig. 8 shows spontaneous extracellular activity from a monolayer of cardiac muscle cells recorded from 4 different FETs. The position of the FETs from which recordings were made is shown in Fig. 8b. All the signals are similar in shape as described in Fig. 3. The amplitude of the signals varies between the different FETs probably because of the variable strength of the coupling. Due to the electrical connections between cells in the layer, the cell with the highest repetition rate of action potentials determines the beating frequency of the whole layer. From the time delay between the recordings of the action potentials at the various sites, the origin, the direction and the velocity (ca 0.2 m/s) of the burst pattern assuming an isotropic spreading of the excitation between the cells can be estimated.

Claims: Claims 1 . An assembly, comprising at least one field-effect transistor comprising a source contact and a drain contact, and one or more layers of vertebrate cells in contact with an non-metalli- zed surface region of the field-effect transistor between the source contact and the drain contact. 2. The assembly according to claim 1 , wherein the vertebrate cells are cells of mammalian origin. 3. The assembly according to claim 1 or 2, wherein the cells are myocardial cells. 4. The assembly according to claim 3, wherein the myocardial cells are beating rat cardiac muscle cells. 5. The assembly according to any one of the claims 1 to 4, wherein the cell density ranges from 10 4 - 1 0 6 cells/cm 2 . 6. The assembly according to any one of the claims 1 to 5, wherein the field-effect transistor is a n- or p-channel silicon field-effect transistor. 7. The assembly according to any one of the claims 1 to 6, wherein the field-effect transistor comprises at least one lll-V or ll-V semiconductor heterostructure. 8. The assembly according to any one of the preceding claims 1 to 7, wherein the size of the non-metallized surface regions of the field- effect transistors ranges from 28x9 μm 2 to 1 0x1 μm 2 . 9. The assembly according to claim 6, wherein the non-metallized surface regions are arranged in a matrix with spaced apart centers, in particular a 4x4 matrix with the centers 200 μm apart. 10. An apparatus for extracellular electrophysiological recordings com- prising the assembly according to any one of the claims 1 to 9, and optionally means for amplifying electrical signals detected at recording terminals of the assembly and/or means for monitoring the electrical signals. 1 1 . An apparatus for extracelluler electrophysiological recordings according to claim 1 0, further comprising a means for measuring time- delays between the electrical signals to determine a burst pattern of signals in the layer/layers of vertebrate cells. 1 2. Use of the assembly according to any one of claims 1 to 9 or the apparatus according to claim 1 0 or 1 1 for testing for testing potential pharmaceutically effective compounds, or compounds having potential side effects, or potentially toxic compounds.

Inventors: KNOLL WOLFGANG, OFFENHAEUSER ANDREAS, KNOLL, WOLFGANG, OFFENHAEUSER, ANDREAS
Applicants: KNOLL WOLFGANG, OFFENHAEUSER ANDREAS, SYMBIOSIS GMBH, KNOLL, WOLFGANG, OFFENHAEUSER, ANDREAS
Application year: 1999
Main classification: G01N27
IPC codes: A61B5, C12M1, C12Q1, G01N27, G01N33