JP2009027942A - Device for cell electrophysiological sensor and cell electrophysiological sensor using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the measurement accuracy of a cell electrophysiological sensor. <P>SOLUTION: This device for the cell electrophysiological sensor has a cell-holding plate 10 having a conducting hole 9, a storage tank 12 disposed on the cell-holding plate 10, and a cell-introducing route 16 for injecting cells in the storage tank 12, wherein the flow exit 18 of the cell-introducing route 16 is upward directed. Thereby, the present invention enables to catch the cells at the opening of the conducting hole 9 more quickly than cell refuses, to improve the adhesiveness of the cells to the opening of the conducting hole 9, and consequently to improve the measurement accuracy of the cell electrophysiological sensor. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor device for measuring a cell response to a drug and the like, and a cell electrophysiological sensor using the device.

  For example, the conventional cell electrophysiological sensor shown in FIG. 8 is provided with a cell holding plate 2 having a conduction hole 1, a storage tank 3 provided above the cell holding plate 2, and a cell holding plate 2 below. A channel 4 and electrodes 5 and 6 disposed in the storage tank 3 and the channel 4 are provided.

  In addition, the storage tank 3 and the flow path 4 are respectively filled with the electrolytic solution, and then the cells 7 are injected into the storage tank 3 using a pipette or the like (not shown). Here, in the cells 7 to be injected, cell debris 8 which is a fragment of the cells 7 is also mixed.

  The potential difference between the electrodes 5 and 6 is measured in a state where the cells 7 are captured and held in close contact with the opening of the conduction hole 1 by suction or the like from the flow path 4 side below the conduction hole 1.

At this time, the potential difference fluctuates by applying a physicochemical stimulus such as administering a drug to the cell 7, so that the electrochemical change of the cell 7 can be analyzed.
JP 2002-518678 Gazette Japanese translation of PCT publication No. 2003-527581

  The conventional device for cell electrophysiology sensor has a problem that the measurement accuracy of the cell electrophysiology sensor is low.

  This is because the adhesion between the cell 7 and the opening of the conduction hole 1 is low.

  That is, the cell debris 8 mixed in the cell 7 may adhere to the periphery of the opening of the conduction hole 1 or may block the opening of the conduction hole 1, and the cell 7 may be held in close contact with the opening of the conduction hole 1. As a result, the measurement accuracy of the cell electrophysiological sensor is lowered.

  Accordingly, an object of the present invention is to improve the measurement accuracy of a cellular electrophysiological sensor.

  In order to achieve this object, the present invention is provided with a cell introduction path for injecting cells into the storage tank, and the cell introduction path has its outlet facing upward.

  Thereby, this invention can improve the measurement precision of a cell electrophysiological sensor.

  This is because the adhesion between the cells and the opening of the conduction hole is improved.

  That is, according to the present invention, since the outlet of the cell introduction path faces upward, the cells and cell debris are injected into the storage tank so as to be blown up, and then fall according to gravity.

  Here, since the cell debris has a more distorted shape than the cells, it is susceptible to the resistance of the liquid, and since its volume is small, the resistance from the liquid is dominant in its falling speed. Therefore, the cell debris falls more slowly than the cell, and the cell is trapped in the opening of the conduction hole earlier than the cell debris.

  As a result, the adhesion between the cells and the opening of the conduction hole is improved, and the measurement accuracy of the cell electrophysiological sensor can be improved.

(Embodiment 1)
As shown in FIG. 1, the cell electrophysiological sensor device according to the present embodiment includes a cell holding plate 10 having a conduction hole 9, a holding plate 11 that supports the outer surface of the cell holding plate 10, the holding plate 11 and A storage tank 12 provided above the cell holding plate 10 and a flow path 13 provided below the holding plate 11 and the cell holding plate 10 are provided. That is, the present embodiment has a structure in which the storage tank 12 and the flow path 13 are partitioned by the cell holding plate 10.

  In this embodiment, the storage tank 12 is formed by a through-hole 15 processed into a well plate 14 (resin substrate), and cell introduction for injecting cells into the storage tank 12 is made inside the well plate 14. A path 16 is formed. The cell introduction path 16 can be formed by injection molding when the well plate 14 is made of resin.

  The cell introduction path 16 is tubular with both ends open, and one opening is exposed on the upper surface of the well plate 14, and this opening serves as a cell inlet 17. The other opening is exposed on the inner wall of the through hole 15 of the well plate 14, and this opening serves as the outlet 18.

  The cell introduction path 16 according to the present embodiment has a structure in which the well plate 14 is temporarily lowered downward from the upper surface of the well plate 14 and then bent obliquely upward to be exposed to the inner wall of the through hole 15 of the well plate 14. . That is, the cell introduction path 16 in the present embodiment has the outflow port 18 facing obliquely upward.

  In this embodiment, the portion of the cell introduction path 16 that is bent upward toward the outlet 18 is inclined upward by about 20 to 30 degrees with respect to the cell holding plate 10. This angle is appropriately adjusted depending on the water pressure at the time of cell injection, the mass of the cell to be examined, and the like.

  In the present embodiment, as shown in FIG. 2, the lower inner wall 19 at the outlet 18 of the cell introduction path 16 is R-processed so as to be curved inward. By curving the outflow port 18 in this manner, it is possible to suppress damage to the good cells that are the subject.

  In the present embodiment, the diameter of the opening of the conduction hole 9 shown in FIG. 1 is 1 μm to 3 μm, and the depth is 3 μm to 10 μm. When the conduction hole 9 having a size in this range is intended for a cell having a diameter of about 10 μm to 100 μm as a subject, the cell can be efficiently adhered and held.

  The cell holding plate 10 of the present embodiment is made of a silicon substrate or a so-called SOI substrate, and can process the fine conduction holes 9 with high accuracy.

  The holding plate 11 was made of epoxy resin or glass, and the well plate 14 was made of epoxy resin or the like.

  The cell electrophysiological sensor using the cell electrophysiological sensor device as described above is one in which the electrodes 20 and 21 are disposed in the storage tank 12 and the flow path 13, respectively. Is not limited to the present embodiment, and an electrolytic solution (intracellular fluid, extracellular fluid, etc. described later) filled in the storage tank 12 above the cell holding plate 10 and the flow path 13 below the cell holding plate 10 is electrically Need only be connected.

  Next, a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor according to the first embodiment will be described with reference to the drawings.

First, the intracellular fluid (electrolytic solution) is filled in the flow path 13 shown in FIG. Here, in the case of mammalian muscle cells, a typical intracellular solution includes an electrolytic solution to which about 4 mM of K ⁺ ions, about 145 mM of Na ⁺ ions, and about 123 mM of Cl ⁻ ions are added.

Thereafter, the extracellular fluid is stored in the storage tank 12. Here, in the case of mammalian muscle cells, typical extracellular fluids include electrolytes to which K ⁺ ions are added at 155 mM, Na ⁺ ions at about 12 mM, and Cl ^- ions at about 4.2 mM. At this time, the extracellular fluid may be injected from other than the cell introduction path 16, but by injecting from the cell introduction path 16, bubbles in the cell introduction path 16 can be expelled to the storage tank 12, and cell injection The outflow of bubbles at the time can be suppressed. Further, the cells can be smoothly injected by pre-wetting the inner wall of the cell introduction path 16.

  Then, when the electrodes 20 and 21 are inserted into the storage tank 12 and the flow path 13 and a measuring instrument (not shown) is connected between the electrodes 20 and 21, the intracellular fluid and cells partitioned by the cell holding plate 10 Electrical characteristics (current, resistance value, voltage, etc.) with the external liquid can be measured.

  In this state, an electrical circuit is formed in which the extracellular fluid and the intracellular fluid are conducted only through the conduction hole 9, and its characteristics are observed as, for example, electrical resistance and IV characteristics, and usually about 1 MΩ. With electrical resistance.

  Next, cells are injected into the storage tank 12 through the cell introduction path 16 in the direction of the arrow 22 in FIG. Since the cells are stored in the extracellular fluid, they are injected together with the extracellular fluid. At this time, the extracellular fluid is injected while being pressurized.

  Then, as shown in FIG. 3, the cells 23 are jetted together with the cell debris 24 from the outlet 18 of the cell introduction path 16 into the storage tank 12. Then, by suctioning the flow path 13 under reduced pressure, the cells 23 are attracted to the conduction holes 9, and the cells 23 block the openings of the conduction holes 9, so that the space between the extracellular fluid and the intracellular fluid is sufficiently high at 1 GΩ or more. Has electrical resistance (called Giga Seal).

  In this giga-seal state, a minute cell membrane hole is opened in the cell membrane of the cell 23 by further increasing the suction pressure.

  As a result, a minute current of about several nA flowing through the ion channel of the cell membrane can be measured, and the electrical characteristics of various ion channels, the response characteristics to drugs, and other responses to external stimuli can be measured.

  Next, the effect in this Embodiment is demonstrated below.

  In the present embodiment, the measurement accuracy of the cell electrophysiological sensor can be improved.

  The reason is that the cells can be efficiently held tightly in the opening of the conduction hole 9.

  That is, conventionally, as shown in FIG. 8, the cells 7 are injected by applying water pressure downward from the upper part of the storage tank 3 toward the cell holding plate 2. Here, at this time, cell debris 8 is inevitably mixed in the cells 7.

  The cell debris 8 may adhere to the periphery of the opening of the conduction hole 1 or may block the opening of the conduction hole 1, and the good cell 7 may not be held in close contact with the opening of the conduction hole 1. It was. In the conventional cell electrophysiological sensor, it is considered that the falling speeds of the cells 7 and the cell debris 8 were greatly influenced by the water pressure at the time of injecting the cells 7 rather than the respective volumes and shapes.

  On the other hand, in this embodiment, as shown in FIG. 3, the cells 23 and the cell debris 24 are injected into the storage tank 12 so as to be once blown up, and then fall according to gravity. Here, since the cell debris 24 has a smaller volume than the cell 23, the drop rate is dominated by resistance from the liquid (extracellular liquid). Accordingly, the cell debris 24 falls more slowly than the cell 23, and the cell 23 is captured in the opening of the conduction hole 9 earlier than the cell debris 24.

  As a result, the cell 23 and the conduction hole 9 can be held in close contact with each other, and the measurement accuracy of the cell electrophysiological sensor can be improved by realizing the above-described giga seal.

  Further, since the cell 23 generally has a larger mass than the cell debris 24, the cell 23 quickly falls downward without being spouted far and reaches the opening of the conduction hole 9 earlier than the cell debris 24.

  Furthermore, since the cell debris 24 has many irregular shapes as compared with the cells 23, the cell debris 24 has a large surface area and is easily subjected to resistance of the liquid (extracellular fluid). Therefore, it takes time to reach the conduction hole 9, and the cell 23 can be captured in the opening of the conduction hole 9 before the conduction hole 9 is blocked by the cell debris 24.

  Further, in the present embodiment, in the bent part of the cell introduction path 16, the flow is faster on the outer periphery than on the inner periphery, so that cells 23 with a large mass gather on the outside and cell debris with a small mass on the inside. 24 is easy to gather.

  Therefore, since the cells 23 are offset toward the lower side of the cell introduction path 16 just before the outlet 18, the lower inner wall 19 of the outlet 18 is curved inward as shown in FIG. By making the shape hang downward, a flow of liquid (extracellular fluid) is formed along the curved surface, and the cell 23 easily falls down along with this flow.

  Furthermore, in the present embodiment, as shown in FIG. 1, the position of the outlet 18 is fixed because the cell introduction path 16 is integrated into the well plate 14. Therefore, it is possible to inject cells while maintaining the optimal position of the outflow port 18, and to efficiently capture the cells.

  In the present embodiment, the opening 18 of the cell introduction path 16 faces obliquely upward, but may be directed vertically upward. In this case, the cell introduction path 16 may be routed from the flow path 13 side to the storage tank 12. As described above, even when the opening 18 of the cell introduction path 16 is directed upward in the vertical direction with respect to the cell holding plate 10, as in the present embodiment, good cells are efficiently captured, and the cell electrophysiological sensor Measurement accuracy can be improved.

(Embodiment 2)
As shown in FIG. 4, the difference between the present embodiment and the first embodiment is that an actuator 25 is arranged on the inner wall of the through hole 15 of the well plate 14 in order to generate a standing wave in the cell introduction path 16. Is a point.

  The actuator 25 in the present embodiment is obtained by sandwiching a piezoelectric layer such as lead zirconate titanate or lithium niobate between two layers of electrodes (such as platinum or gold), and generates a standing wave in the piezoelectric layer. Therefore, for example, a voltage that generates vibration with the frequency f below is applied.

That is, the frequency f is the width of the cell introduction path 16 W, and the sound velocity in the liquid component of the liquid component (extracellular fluid) and solid components (cells, cell debris) introduced into the cell introduction path 16 is v. Then f = (n / 2) × v / W (n is a natural number)
The frequency f satisfies

  As a result, a standing wave is generated in the cell introduction path 16, and as shown in FIG. 5, one node 26 is formed in the center of the cell introduction path 16, and the cells 23 can be aligned in the node 26.

  Since the cell 23 generally has a larger volume than the cell debris 24, the magnitude of the force received from the standing wave is large, and the cells 23 can be preferentially collected in the nodes 26.

  If the cells 23 are aligned in the cell introduction path 16 as described above, the cells 23 are directed toward the opening of the conduction hole 9 by spraying the cells 23 into the storage tank (12 in FIG. 4) at a constant water pressure. Can be dropped in place. Therefore, the capture rate of the cells 23 can be improved, and the measurement accuracy of the cell electrophysiological sensor can be improved.

  In the present embodiment, as shown in FIG. 5, a standing wave is generated in the cell introduction path 16 and the cells 23 are aligned, but the standing wave vibration may not be used. That is, when traveling wave vibration is generated in the cell introduction path 16, it is possible to suppress the cells 23 and the cell debris 24 from sticking into the cell introduction path 16 due to the vibration.

  In addition, for example, by providing a plurality of actuators (25 in FIG. 4) and increasing or decreasing the voltage applied to these actuators 25, the cells 23 that are relatively large in volume and easy to move, and the cell debris 24 that is small in volume and difficult to move Can be separated. Therefore, the cells 23 and the cell debris 24 can be sprayed into the storage tank (12 in FIG. 4) in a separated state, and only the cells 23 are easily captured in the conduction holes (9 in FIG. 4).

  Description of other configurations and effects similar to those of the first embodiment is omitted.

(Embodiment 3)
The difference between the present embodiment and the first embodiment is that, as shown in FIG. 6, a tubular cell introduction path 16 is attached to the inner wall of the through hole 15 of the well plate 14, and the shape of this cell introduction path 16 is obliquely upward. It is the point made into the spiral which rotates diagonally downward from.

  As shown in FIG. 7, in the portion where the cell introduction path 16 rotates in a spiral manner, the cell 23 having a large mass is moved outside the cell introduction path 16 by the centrifugal force in the direction of the arrow 27, as shown in FIG. 7. The waste 24 is shifted to the inside, and at the outlet 18, the cells 23 are gathered downward and the cellular waste 24 is gathered upward.

  In this state, since the cells 23 and the cell debris 24 are injected into the storage tank (12 in FIG. 6), it is considered that the cell debris 24 is blown upward. Further, the cell 23 falls downward and is quickly captured by the opening of the conduction hole 9, so that the measurement accuracy of the cell electrophysiological sensor can be improved.

  Description of other configurations and effects similar to those of the first embodiment is omitted.

  The present invention can efficiently capture cells in the opening of the conduction hole and is effective in improving the measurement accuracy of the cell electrophysiological sensor.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention X section enlarged sectional view of FIG. The principal part expanded sectional view which shows operation | movement of the cell electrophysiological sensor in Embodiment 1 of this invention. Sectional drawing of the cell electrophysiological sensor in Embodiment 2 of this invention Sectional drawing which shows operation | movement of the same cell electrophysiological sensor typically Sectional drawing of the cell electrophysiological sensor in Embodiment 3 of this invention The principal part expanded sectional view which shows operation | movement of the cell electrophysiological sensor Sectional view of a conventional cellular electrophysiological sensor

Explanation of symbols

DESCRIPTION OF SYMBOLS 9 Conduction hole 10 Cell holding plate 11 Holding plate 12 Reservoir 13 Flow path 14 Well plate 15 Through hole 16 Cell introduction path 17 Inlet 18 Outlet 19 Inner wall 20 Electrode 21 Electrode 22 Arrow 23 Cell 24 Cell debris 25 Actuator 26 Node 27 Arrow

Claims (6)

A cell holding plate having a conduction hole;
A storage tank provided above the cell holding plate;
A cell introduction path for injecting cells into this reservoir,
This cell introduction path is a device for a cell electrophysiological sensor in which the outflow port faces upward. The storage tank is
Formed with through holes provided in the substrate,
The cell introduction path is
The cell electrophysiological sensor device according to claim 1, which is formed inside the substrate. The device for cell electrophysiological sensors according to claim 1, further comprising an actuator for applying vibration in the cell introduction path. The cell electrophysiological sensor device according to any one of claims 1 to 3, wherein the cell introduction path has a spiral shape that rotates in a vertical direction. The device for cell electrophysiological sensors according to any one of claims 1 to 4, wherein an inner wall below the outlet of the cell introduction path is curved inward. A device for a cellular electrophysiological sensor according to any one of claims 1 to 5,
A cell electrophysiological sensor comprising an electrolytic solution injected above and below the cell holding plate of the device for cell electrophysiological sensor and electrodes electrically connected to each other.

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