Method and apparatus for performing automated patch-clamp analysis on multiple living cells.

Abstract

A method and apparatus for automated patch-clamp analysis of multiple living cells clamped in each patch-clamp well. At least one cell enters voltage-clamp mode by applying a defined voltage to it. The voltages of all cells may be the same. While at least one cell is in voltage-clamp mode, the values ​​of the parameters associated with voltage-clamp mode are determined, and a transition from voltage-clamp mode to current-clamp mode is performed. In current-clamp mode, a defined current is applied to the cells. The defined current can be set individually for each cell. The transition from voltage-clamp mode to current-clamp mode and / or the characteristics of current-clamp mode are controlled based on the parameter values ​​determined while the cell was in voltage-clamp mode.

Description

Technical Field

[0001] The present invention relates to a method and apparatus for automatically performing patch-clamp analysis on a plurality of living cells, particularly cells that generate action potentials. More specifically, the present invention relates to a method and apparatus for placing a cell in current-clamp mode, wherein changes in transmembrane voltage due to ion-channel activity are detected. With this technique, a researcher can control the amount of current applied to a cell and thereby control the transmembrane potential.

Background Art

[0002] Ion channels are transmembrane proteins that catalyze the transport of inorganic ions across cell membranes. Ion channels are involved in various processes such as the generation and timing of action potentials, synaptic transmission, hormone secretion, and muscle contraction. Many drugs exert specific effects through the regulation of ion channels. Patch-clamp analysis is a well-known technique for modulating chemically induced ion-channel activity.

[0003] The originally developed patch-clamp method has achieved great development in the fields of biology and medicine. This is because this technique enables the measurement of ion currents passing through a single ion channel and the study of the response of ion channels to drugs. Ion channels are membrane proteins present inside the cells of organisms. The flow of ions in and out of cells is controlled by the membrane, and ion channels have become important targets for various drugs.

[0004] In both single-channel recording and whole-cell recording, the activity of individual channel subtypes can be characterized by imposing a "voltage clamp" on the entire membrane. In the voltage-clamp method, membrane current is recorded at a defined membrane potential. In the current-clamp method, changes in transmembrane voltage due to ion-channel activity are detected, and a defined current is applied to the cell to trigger a cell action potential that enables the measurement of transmembrane potential in current-clamp mode.

[0005] Measurement protocols typically specify pressure, potential, or current, which is applied to the patch-clamp site to measure current, capacitance, or cell potential across the ion channel. Patch-clamp amplifiers are ubiquitous tools for characterizing ion channel activity.

[0006] Current patch-clamp methods and equipment have drawbacks. Existing methods and equipment for performing patch-clamp analysis on multiple cells have limitations. Furthermore, it has been found that transitioning from voltage-clamp mode to current-clamp mode causes the patch-clamp amplifier to clip due to transient phenomena, reducing the success rate of current-clamp analysis.

[0007] Given the above background, the objective of the embodiments of the present invention is to improve the success rate of patch-clamp analysis, particularly analysis in current-clamp mode, and in particular to reduce the occurrence of transient phenomena when transitioning from voltage-clamp mode to current-clamp mode. [Overview of the project]

[0008] In a first embodiment, the present invention provides a method for performing automated patch-clamp analysis on a plurality of living cells, wherein each of the cells is patch-clamped in a patch-clamp device comprising a plurality of patch-clamp wells, and the method independently and in parallel for each of the cells, the following steps: Clamp each cell into its respective well, To put at least one cell, for example, each cell, into voltage clamp mode by applying a defined voltage or voltage protocol to each of at least one cell, To determine the values ​​of parameters related to voltage clamp mode while at least one cell is in voltage clamp mode, Transitioning from voltage clamp mode to current clamp mode while applying a predetermined current to at least one cell, or transitioning from voltage clamp mode to current clamp mode and then applying a predetermined current to at least one cell while in current clamp mode, Controlling the characteristics of the transition and / or current clamp mode based on the parameter values ​​determined while at least one cell is in voltage clamp mode, A method for providing this.

[0009] In a second embodiment, the present invention provides an apparatus for performing automated patch-clamp analysis on a plurality of living cells, the apparatus comprising: The device comprises at least one patch-clamp well, for example, multiple patch-clamp wells for clamping one of each of the cells, and the device is configured to put at least one of the cells into voltage-clamp mode by applying a defined voltage or voltage protocol to one of each of the at least one of the cells, The aforementioned device further, A sensor system configured to determine the values ​​of parameters associated with voltage clamp mode while at least one cell is in voltage clamp mode, For at least one well or one of each well, independently and in parallel, This causes a transition from voltage clamp mode to current clamp mode. While the cells are in current-clamp mode, a predetermined current is applied to at least one cell. The characteristics of the transition and / or current clamp mode are controlled based on the parameter values ​​determined while at least one cell is in voltage clamp mode. A controller configured as follows, A device equipped with the following features.

[0010] For example, the method may include controlling the characteristics of the transition and / or current-clamp mode based on one or more parameters determined while at least one cell is in voltage-clamp mode.

[0011] It has been found that transient phenomena during the transition from voltage clamp mode to current clamp mode can be reduced by clamping the cells to the predicted voltage before transitioning to current clamp mode. In particular, an improvement in success rate has been demonstrated.

[0012] In one embodiment of the present invention, while each cell is in voltage clamp mode, the value of the parameter associated with voltage clamp mode is a selected current value, and based on this, the holding current required to hold the cell at a defined voltage in current clamp mode is determined. That is, the step of controlling the transition and / or characteristics of current clamp mode is performed based on the selected current value. The magnitude of the holding current for each cell constitutes or is included in the parameter value on which the transition and / or characteristics of current clamp mode are controlled. In particular, the magnitude of the holding current for each cell can be set as the respective defined current in current clamp mode.

[0013] In one embodiment, in voltage clamp mode, cells are clamped in each well with the same defined voltage or voltage protocol. For example, if the same type of cells are clamped in the wells, it is advantageous that the same voltage can be applied in voltage clamp mode. The voltage applied in voltage clamp mode is, for example, the resting membrane potential of the cell. The resting membrane potential, here, is the potential measured when no current is flowing. In other embodiments of the present invention, cells can be clamped in each well with individually set voltages in voltage clamp mode to account for different cell attributes or different cell types in the wells, such as different sizes or different cell resistance seals. The voltage applied in voltage clamp mode may be the resting membrane potential. Instead of applying a defined voltage, a “voltage protocol” (a predetermined set of voltages) can also be applied to each cell.

[0014] All living cells in multiple wells may be of the same type. For example, all living cells in all wells may be action potential-producing cells such as cardiomyocytes or nerve cells. Alternatively, different types of cells may be placed in different wells simultaneously. The patch clamp parameters for each type of cell can be controlled individually using voltage clamp mode and current clamp mode, respectively.

[0015] As a similar example, in current clamp mode, the current applied to each cell may be defined individually for each cell to take into account different cell attributes, or a common value may be set for all cells.

[0016] According to the present invention, the transition and / or characteristics of the current clamp mode are controlled based on the values ​​of the respective parameters determined while each cell is in the voltage clamp mode. The characteristics of the current clamp mode are appropriately controlled based on the voltage maintained in each cell. In one embodiment, the parameter value can be the holding current required to hold the cell at a defined voltage in the voltage clamp mode. For example, the defined voltage may be the resting membrane potential or any other desired potential. To suppress the occurrence of transient phenomena, the current in the current clamp mode is initially set to the same value as the holding current and may, in some cases, be adjusted at a later point to avoid membrane potential drift, etc.

[0017] The transition from voltage clamp mode to current clamp mode and / or the characteristics of current clamp mode can, alternatively or additionally, be determined based on the difference between the measured current in voltage clamp mode and the target current in current clamp mode. The measured current may be the “selected current value” specified above. In most embodiments of the present invention, it is preferable that the difference between the measured current and the target current be as small as possible, but in voltage clamp mode, differences may occur as a result of temporal fluctuations that cannot be fully compensated for, inaccuracies in measurement accuracy, changes in external conditions, etc. For example, the value of the current applied to each cell can be determined based on a term proportional to the difference between the holding current in voltage clamp mode and the target current in current clamp mode. Alternatively, the value of the voltage applied to each cell may be determined based on a term proportional to the change in voltage across each cell.

[0018] The defined current applied to each cell in current clamp mode can be controlled based on a predetermined protocol that defines the pulse width and / or the pulse amplitude of the current pulse. A common control can be applied to all cells, or individual cells can be targeted with individual protocols. For example, since individual cells may have different firing thresholds, individual protocols can be applied for individual cells in order to find the correct pacing stimulus. The charge applied through the pulsed current can be controlled by varying the pulse width and / or the pulse amplitude for each cell. In one embodiment, the current required to induce an action potential in each cell is determined, and the current amplitude applied in current clamp mode is set to be at least equal to the determined current. Preferably, the current amplitude can be set to an amount that exceeds the determined current by, for example, 50%.

[0019] The defined current applied to each cell in current clamp mode can be varied over time to keep the parameters of each cell at a defined level. In particular, the current applied to each cell in current clamp mode can be controlled so that the membrane potential becomes constant or remains within a predefined range.

[0020] The measurement of the values of the parameters associated with voltage clamp mode, and / or further measurements in one of the individual cells, can preferably be performed repeatedly over a certain period rather than at a single point in time. This can suppress the occurrence of transient phenomena when transitioning from voltage clamp mode to current clamp mode, and furthermore, the risk that the membrane potential drifts from the intended potential can be effectively reduced.

Brief Description of the Drawings

[0021] [Figure 1] Figure 1 shows the membrane potential of a cell in a method that does not monitor the membrane potential. [Figure 2] Figure 2 shows the membrane potential of a cell in a method that monitors the membrane potential. [Figure 3]Figure 3 shows the improvement of the action potential phenotype in cardiomyocyte - shaped human induced pluripotent stem cells (hiPSCs) achieved by an embodiment of the present invention. [Figure 4] Figure 4 shows the improvement of the action potential phenotype in cardiomyocyte - shaped human induced pluripotent stem cells (hiPSCs) achieved by an embodiment of the present invention. [Figure 5] Figure 5 is a flowchart showing an embodiment of the method according to an embodiment of the present invention.

Mode for Carrying Out the Invention

[0022] Figure 5 shows a flowchart of the method according to an embodiment of the present invention. In step S100, one or more living cells are clamped in each or some of a plurality of wells of a patch - clamp device. In step S102, each cell is put into voltage - clamp mode by applying a defined voltage or voltage protocol to each cell. In step S104, while each cell is in voltage - clamp mode, the value of each parameter related to the voltage - clamp mode is determined. The transition from voltage - clamp mode to current - clamp mode is performed in step S106 while applying a determined current to each cell. Alternatively, the transition from voltage - clamp mode to current - clamp mode follows step S108 - while the cell is in current - clamp mode, applying a defined current to each cell. In step S110, based on the value of each parameter determined while each cell is in voltage - clamp mode, the transition and / or characteristics of the current - clamp mode are controlled.

[0023] (Example 1) HEK cells were placed in a 24×16 - well two - dimensional array and transferred from voltage - clamp to current - clamp mode to examine the change of Kv1.3 channels.

[0024] In the first approach, a voltage of -90mV was applied to the cells in voltage clamp mode. The transition from voltage clamp mode to current clamp mode was performed by first controlling the current to zero. Of the 384 wells, the amplifier clipped 212 wells.

[0025] In the second approach, the resting membrane potential in voltage-clamp mode was initially determined to be -30mV at I=0. The transition from voltage-clamp mode to current-clamp mode was initiated when it was confirmed that -30mV had been applied to each cell. Of the 384 wells, the amplifier clipped 90 wells.

[0026] This example demonstrates that by setting the voltage in voltage clamp mode to the resting membrane potential and then controlling the transition from voltage clamp mode to current clamp mode, the occurrence of amplifier clipping due to transient phenomena was suppressed.

[0027] (Example 2) In a mimicry-adaptive protocol observing one cell at a time, the current was measured in a voltage-clamp mode at -70mV during the first protocol. Then, the second protocol was performed, initially clamping the cell to -70mV in voltage-clamp mode, and then transitioning to current-clamp mode while clamping the cell with the current measured in the first protocol. This operation was repeated 384 times for 384 cells.

[0028] As a result, the membrane potential was -70mV, but this was only observed in one cell out of 384 cells, which had been measured previously.

[0029] This procedure was repeated 384 times for 384 cells. The relevant potential was measured for each cell or multiple cells in a single well.

[0030] (Example 3) In the first approach, a transition from voltage-clamp mode to current-clamp mode was performed in a single cell. The membrane potential of the entire cell was not monitored. The membrane potential drifted from approximately -30mV to approximately 0mV within approximately 12 seconds, as shown in Figure 1.

[0031] As a second approach, a transition from voltage-clamp mode to current-clamp mode was performed in a single cell. The membrane potential of the entire cell was monitored, and the current applied to the cell was controlled so that the membrane potential remained substantially constant. As shown in Figure 2, no current flowed through the membrane.

[0032] (Example 4) Figure 3 shows the improvement in the action potential phenotype in human induced pluripotent stem cells (hiPSCs) with cardiomyocyte morphology achieved by embodiments of the present invention. In Figure 3, “Cell 2” represents a cell expressing the ion channels necessary to bring the resting membrane potential closer to the expected level. As shown, the voltage measurements in a standard (i.e., prior art) current-clamp ("CC") procedure are equivalent to the voltage measurements in the procedure according to embodiments of the present invention ("adapted CC"), and the measured voltage in current-clamp mode asymptotically converges toward approximately -70 mV.

[0033] In Figure 3, "Cell 3" represents a cell with an elevated resting membrane potential. In the standard procedure, the membrane potential in current-clamp mode asymptotically converges to approximately -45 mV. The procedure according to the embodiment of the present invention resets otherwise inactivated Nav channels, and as shown in Figure 3, the membrane potential in current-clamp mode asymptotically converges towards approximately -70 mV. Thus, the cell can initiate a physiologically more appropriate action potential in the method according to the embodiment of the present invention ("adaptive CC") than in the method according to the prior art ("standard CC").

[0034] (Example 5) Figure 4 shows an improvement over the standard, i.e., prior art procedure ("standard CC"), of the action potential phenotype in human induced pluripotent stem cells (hiPSCs) with cardiomyocyte morphology achieved by an embodiment of the present invention ("adaptive CC").

[0035] As shown, when the embodiments of the present invention are applied, all extracted parameters become more reproducible across cells. This improvement is expected to enhance the throughput of action potential measurements in hiPSC CMs and neurons.

Claims

1. A method for performing automated patch-clamp analysis on multiple living cells, wherein each cell is patch-clamped in a patch-clamp device having multiple patch-clamp wells, and the following steps are performed independently and in parallel for each well: To fix each cell in its respective well, Setting at least one cell to voltage clamp mode by applying a defined voltage or voltage protocol to each of at least one cell, To determine the values ​​of the parameters associated with voltage clamp mode while at least one cell is in voltage clamp mode, Transitioning from voltage clamp mode to current clamp mode while applying a defined current to each of at least one cell, or transitioning from voltage clamp mode to current clamp mode while the cell is in current clamp mode, and then applying a defined current to each of at least one cell, Controlling the characteristics of the transition and / or current clamp mode based on the parameter values ​​determined while at least one cell is in voltage clamp mode, A method for providing this.

2. In the aforementioned voltage clamp mode, all cells in each well are clamped with the same specified voltage. The method according to claim 1.

3. In the current clamp mode, the specified current applied to each cell is defined individually for each of the multiple cells. The method according to claim 1 or claim 2.

4. The value of the parameter associated with the voltage clamp mode while each cell is in the voltage clamp mode is a selected current value, and based on this current value, the holding current required to hold the cell at a specified voltage in the current clamp mode is determined. The method according to any one of claims 1 to 3.

5. The characteristics of the current clamp mode are controlled based on the respective voltages to be maintained for each cell. The method according to any one of claims 1 to 4.

6. The transition from the voltage clamp mode to the current clamp mode and / or the characteristics of the current clamp mode are determined based on the difference between the current measured in the voltage clamp mode and the target current in the current clamp mode. The method according to claim 5.

7. The value of the current applied to each cell is determined based on a term proportional to the difference between the holding current in the voltage clamp mode and the target current in the current clamp mode. The method according to claim 6.

8. The voltage applied to each cell is determined based on a term that is proportional to the change in voltage across each cell. The method according to claim 6.

9. In the current clamp mode, the specified current applied to each cell is controlled based on a predetermined protocol that defines the pulse width and / or pulse amplitude of the current pulse. The method according to any one of claims 1 to 8.

10. In the current clamp mode, the specified current applied to each cell changes over time in order to maintain the parameters of each cell at a specified level. The method according to any one of claims 1 to 9.

11. The parameter for each cell is the resting membrane potential of each cell. The method according to claim 10.

12. All living cells in all wells are cells that generate action potentials. The method according to any one of claims 1 to 11.

13. A device that performs automated patch-clamp analysis on multiple living cells, Each cell has at least one patch clamp well for clamping, wherein the device is configured to put at least one cell into voltage clamp mode by applying a defined voltage or voltage protocol to each of the at least one cell. A sensor system configured to determine the value of a parameter associated with the voltage clamp mode while at least one cell is in the voltage clamp mode, A controller configured to independently and in parallel perform the following operations for each of at least one well, Equipped with, The aforementioned controller, This causes a transition from voltage clamp mode to current clamp mode. While the cells are in current-clamp mode, a defined current is applied to each of the at least one of the cells. Based on the parameter values ​​determined while at least one cell is in voltage clamp mode, the characteristics of the transition and / or the current clamp mode are controlled. Device.

14. The sensor system is configured to measure the values ​​of parameters related to the voltage clamp mode and / or perform further individual measurements of each cell. The apparatus according to claim 13.

15. The sensor system is configured to perform measurements repeatedly over a certain period of time. The apparatus according to claim 13.

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