JP2007124971A - Vessel, method for immobilizing cell in the vessel, method for measurement of intracellular molecular dynamics of cell immobilized by the method and apparatus for measuring intracellular molecular dynamics - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vessel capable of immobilizing a live cell and suitable for the analysis by fluorescence correlation spectroscopy, etc., a method for immobilizing a cell in the vessel, a method for the measurement of intracellular molecular dynamics of the cell immobilized by the method, and an apparatus for the measurement of intracellular molecular dynamics. <P>SOLUTION: The vessel has a cell-holding part having a circular flat bottom of ≥2μm diameter, capable of holding a cell, and having a tapered side wall. The vessel has a flat bottom, and the cell-holding part forms the bottom of a specimen holding part to receive and hold a liquid containing cells. The apparatus for the measurement of intracellular molecular dynamics is provided with the vessel, a cell-transfer means to apply at least one kind of force selected from vibration, pressure, centrifugal force, electric field and magnetic field to the cells in the specimen holding part, and a means to detect the signal of a labeled cell. A cell in the specimen holding part is transferred and immobilized to the cell-holding part by the cell transfer means and the signal of the cell is detected by the signal detecting means. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a container for immobilizing a cell, a method for immobilizing a cell in the container, a method for immobilizing a cell in the container, and a method for immobilizing the cell in the container, which are suitable for measuring the dynamics of the labeled molecule in the cell by incorporating the labeled molecule into the cell. The present invention relates to a method for measuring intracellular molecular dynamics and an apparatus for measuring intracellular molecular dynamics.

  Currently, as methods for analyzing intracellular molecular dynamics, Fluorescence Correlation Spectroscopy (hereinafter abbreviated as FCS), Fluorescence Intensity Distribution Analysis (hereinafter abbreviated as FIDA), and the like are known. ing. These are techniques in which a molecule labeled with a fluorescent substance is taken into a cell to be observed and the dynamics of the labeled molecule in the cell is detected as fluctuations in the fluorescence signal. In addition, by this method, a plurality of cells can be observed at the same time to analyze the exchange of molecules due to tight junctions between these cells.

In the analysis method, one or a plurality of cells to be observed are held in a container, and the fluorescence signal is detected by irradiating the cells with light having a wavelength capable of exciting the fluorescent substance. Is required to accurately detect molecular dynamics such as the intensity and movement speed of a fluorescent signal by confocaling a very narrow specific region in a cell.
Conventionally, as a container used for the measurement of such a fluorescent signal, a container having a bottom area of about 0.8 square centimeter in which eight chambers are occupied by a cover slip, a diameter of 0.8 centimeter, and a bottom area of 0.5 square centimeter are used. A multi-well glass bottom plate or the like is used.
Here, in order to accurately analyze the molecular dynamics in the cell, it is preferable to use a living cell. In this case, the cell to be observed is placed on the container as described above or a commonly used microplate for observation. When trying to do so, the cells move during the measurement, so that there is a problem that the molecular dynamics cannot be detected accurately. That is, in analysis such as FCS and FIDA, it is necessary to fix living cells so as not to move.

In order to solve this problem, as a method for fixing cells to be observed, for example, a method using a microplate having small wells for capturing cells is disclosed (for example, Patent Document 1). And 2).
In addition, a cell having a suction port on the bottom surface is used to capture one cell having a size larger than the suction port, and the cell is sucked through the suction port to open a hole in the cell. A method for fixing cells in the cell (patch clamp method) is disclosed. The patch clamp method is a method suitably used for measuring a response response to a drug by a change in current in a single observation target cell.
International Publication No. 2004/077009 Pamphlet International Publication No. 2004/113492 Pamphlet International Publication No. 2005/007866 Pamphlet

  However, in the inventions described in Patent Documents 1 and 2, even if the cells can be trapped in the well, there are cells of various sizes, and each time the size of the well is changed to the size of these various cells. There is a problem that it is difficult to make it suitable. That is, if the size of the well is too large relative to the size of the cell, even if one or more cells can be captured in the well, it cannot be firmly held in the well.

  On the other hand, the invention described in Patent Document 3 is suitable for measuring changes in intracellular potential and the like because a suction port is provided on the bottom surface of the container, but it is desired to detect a fluorescent signal with high accuracy. In some cases, it is not preferable to irradiate light for exciting the fluorescent substance from the bottom side of the container, and it is necessary to irradiate from above the cells. However, even if an attempt is made to detect a fluorescent signal by irradiating light from above the cell in this way, there is a problem that a lot of noise is picked up and measurement with high accuracy becomes impossible. In addition, since the suction port is small, only one cell can be captured in the container, and there is a problem that tight junctions between a plurality of cells cannot be measured. Furthermore, since cells are perforated, there is a problem that accurate molecular dynamics in cells cannot always be analyzed.

  The present invention has been made in view of the above-described problems, and is suitable for analysis such as fluorescence correlation spectroscopy (FCS), which can fix a living cell to be observed, and the cell in the container. It is an object of the present invention to provide a fixing method, a method for measuring intracellular molecular dynamics of a cell fixed by the method, and an apparatus for measuring intracellular molecular dynamics.

To solve the above problem,
The invention according to claim 1 is a container having a cell holding portion having a circular bottom surface with a diameter of at least 2 μm, a shape on which cells can be placed and flat, and a side surface having a tapered shape. Is flat, and the cell holding part is a bottom part of a sample holding part for adding and holding a liquid containing cells.

  The invention according to claim 2 is the container according to claim 1, wherein the bottom surface of the cell holding portion is a circle having a diameter of 2 μm or more.

  Invention of Claim 3 is a container of Claim 1 or 2 characterized by the height of the said cell holding | maintenance part being 4 micrometers or more.

  The invention according to claim 4 is the container according to any one of claims 1 to 3, wherein the entire side surface of the sample holder is tapered.

  The invention according to claim 5 is characterized in that one end of the flow path leading to the outside of the container is opened on the side surface in the vicinity of the bottom surface of the cell holding part. The container.

  Invention of Claim 6 consists of a material with a refractive index of 1.5 or less, It is a container as described in any one of Claims 1-5 characterized by the above-mentioned.

  The invention according to claim 7 is characterized in that the side surface of the sample holder other than the cell holder is made of a material having a contact angle with water of 100 ° or more. The container according to one item.

  Invention of Claim 8 is a container as described in any one of Claims 1-7 by which the side surface of the said cell holding part is colored black.

  The invention according to claim 9 is a container having a plurality of the sample holders, wherein the distance between the centers of the bottom surfaces of the cell holders adjacent to each other is 1 mm or more. It is a container given in any 1 paragraph.

  Invention of Claim 10 adds the liquid containing the cell which took in the label | marker molecule | numerator to the sample holding part of the container as described in any one of Claims 1-9, vibration, pressure is applied to a cell. The cell fixing method is characterized by adding at least one selected from the group consisting of gravity, centrifugal force, and electrostatic attraction, and moving and fixing the cell to the cell holding part.

  The invention according to claim 11 adds a liquid containing cells into which the labeled molecules and magnetic particles have been incorporated into the sample holding part of the container according to any one of claims 1 to 9, so that the magnetic force is applied to the cells. Is applied to the cells, and the cells are moved to the cell holding part and fixed.

  The invention according to claim 12 is the cell fixing method according to claim 10 or 11, wherein the labeling molecule is a molecule labeled with a fluorescent substance.

  The invention according to claim 13 is to detect the signal of the labeled molecule in the cell fixed by the cell fixing method according to any one of claims 10 to 12 from the bottom side of the cell holding part. This is a characteristic method for measuring intracellular molecular dynamics.

  The invention according to claim 14 is selected from the group consisting of vibration, pressure, centrifugal force, electric field, and magnetic force for the container according to any one of claims 1 to 9 and the cells in the sample holder of the container. A device for measuring intracellular molecular dynamics, characterized by comprising: a cell moving means for adding at least one of the above and a signal detecting means for detecting a signal of a labeled molecule in the cell.

  The invention according to claim 15 has a sample addition means for adding a liquid containing cells into which a labeled molecule has been incorporated into the sample holding part of the container. It is a measuring device for dynamics.

  The invention according to claim 16 is the device for measuring intracellular molecular dynamics according to claim 14 or 15, wherein the labeling molecule is a molecule labeled with a fluorescent substance.

  According to the present invention, since one or a plurality of living cells can be fixed in a container, molecular dynamics in a cell using optical means, which has been difficult to measure conventionally due to cell movement. Can be measured easily and with high accuracy. For example, by applying a technique such as FCS or FIDA, it is possible to measure tight junctions between a plurality of living cells, which has been difficult in the past. In addition, by screening drug discovery candidate substances that have been screened by IN VITRO such as electrophoresis and ELISA using living cells, reactions close to in vivo can be observed.

Hereinafter, the present invention will be described in detail.
The container of the present invention is a container having a cell holding portion having a circular shape with a bottom surface of at least 2 μm in diameter, capable of placing cells, and having a flat side surface and a tapered side surface, and the bottom surface of the container is flat. In addition, the cell holding part is a bottom part of a sample holding part for adding and holding a liquid containing cells.

  In the present invention, in order to accurately perform optical measurement such as detection of a signal in a cell to be measured, the light irradiation surface, the container corresponding to the signal detection surface, and the bottom surface of the cell holding portion are flat. .

  Further, the container of the present invention needs to be able to hold at least one cell of various sizes in its cell holding part. Since the normal cells to be observed are the smallest and have a size of about 5 μm, the bottom surface of the cell holding part is almost sufficient if it has a shape on which a circular one having a diameter of 5 μm can be placed. However, as a special cell, platelets have a size as small as 2 to 3 μm. Therefore, in the container of the present invention, the bottom surface of the cell holding part has a shape on which a circular one having a diameter of at least 2 μm can be placed. ing. Among these, a circular shape having a diameter of 2 μm or more is preferable.

  Further, it is not always necessary to fix the cells to be observed so as to be in contact with the bottom surface of the cell holding portion. Even if the cell is not in contact with the bottom surface of the cell holding part, the cell is fixed by being in contact with the side surface.

On the other hand, by making the side surface of the cell holder tapered, one or more cells can be held on the cells fixed to the bottom of the cell holder, and the tight junction between cells can be measured. Can do.
The size of platelets is 2 to 3 μm, the size of animal cells is about 5 to 50 μm, and the size of plant cells is about 10 to 500 μm. In view of this, in consideration of stacking and fixing animal cells that are particularly mobile so that two or more cells can be stacked on the cell holding part, the height of the cell holding part is preferably 4 μm or more, and preferably 10 μm or more. It is more preferable that it is 100 μm or more.

  Further, the side surface of the cell holding part may have a shape with a constant taper angle or a shape with a different taper angle, and is not particularly limited, but the taper angle is 90 ° with respect to the bottom surface of the cell holding part. It is preferably not less than 170 degrees and not more than 170 degrees, more preferably not less than 110 degrees and not more than 145 degrees, and further preferably not less than 120 degrees and not more than 135 degrees. This taper angle can be selected depending on the ease of movement of the cells to the cell holding portion and the size of the cells to be fixed. For example, when cells are floated from the bottom surface of the cell holding part and supported and fixed on the side surfaces, the taper angle is set so that the distance between the cell to be fixed and the flat bottom surface of the cell holding part is 50 μm to 200 μm. If selected, the distance between the cell and the bottom of the cell holder is not too close, and mixing of the solution around the cell is not hindered when a drug is added, etc., so the solution is sufficiently mixed and the biological reaction is accurately detected can do.

  Although the sample holding part of the container of the present invention has a cell holding part having a tapered side surface at the bottom, the shape of the side face other than the cell holding part is not particularly limited. However, after adding a cell-containing solution to the sample holder, the cells can be easily moved to the cell holder simply by applying a small force to the cells, so the entire side surface of the sample holder is tapered. It is preferable that

The cell holding part of the container of the present invention may be one in which one end of a flow path leading to the outside of the container is opened on the side surface near the bottom surface. By using a container having a cell holding part having such a shape, a liquid containing cells is added to the sample holding part, and then a pump is connected to the other end of the flow path to decompress the inside of the cell holding part. The cells can be easily moved to the cell holding part.
The shape and length of the flow path are not particularly limited, but the area of the opening in the cell holding part needs to be smaller than the size of the cell.
Moreover, if one end of the flow path opened in the cell holding part is provided at the bottom of the cell holding part, the molecular dynamics in the cell cannot be measured accurately, and if it is provided on the upper side of the cell holding part, Since it becomes difficult to move cells by decompression, it is preferable to provide the cells on the side surface near the bottom surface of the cell holding portion as described above.
Furthermore, the number of flow paths provided in one cell holding part is not particularly limited, and a plurality of flow paths may be provided, but a single cell can provide a sufficient effect of moving cells to the cell holding part.

The number of sample holders included in the container of the present invention is not particularly limited, and may be appropriately selected according to the purpose of measurement.
In the case of a container having a plurality of sample holders, it is preferable to provide a certain distance between the respective cell holders according to the numerical aperture of the objective lens of the detector used for signal measurement. For example, when the numerical aperture is about 0.9 to 1.2, the distance between the centers of the bottom surfaces of the cell holding portions adjacent to each other is preferably 1 mm or more, and more preferably 3 to 5 mm. By doing in this way, the influence of the noise light from the adjacent cell holding part at the time of signal measurement can be reduced, and the molecular dynamics in the cell can be measured with higher accuracy.

  The material of the container of the present invention is not particularly limited as long as the signal in the measurement target cell can be measured with high accuracy, but light scattering can be reduced and noise can be reduced, so that the refractive index is 1. It is preferable that the material is 5 or less. As such, for example, glass having a refractive index of 1.5 or less, or a fluororesin such as tetrafluoroethylene (refractive index 1.35), silicon resin (refractive index 1.43), polymethylpentene (refractive index). 1.463), butyl butyral (refractive index 1.47), polyacetal (refractive index 1.48), PMMA (refractive index 1.488), polypropylene (refractive index 1.49), polyallomer (refractive index 1.492) ) And the like.

Moreover, you may use the kind of material of a container which is different in the bottom face and side surface of a cell holding part. That is, when the molecular dynamics in the cell is measured from the bottom surface side, the bottom surface of the cell holding part is made of a material having a refractive index of 1.5 or less and the side surface is made of a material having a smaller refractive index. Furthermore, light scattering can be suppressed and noise can be reduced.
The combination of materials used for the bottom and side surfaces of the cell holding part is preferably selected from the above, but the refractive index of the liquid containing the cells to be measured added to the sample holding part is selected from the materials used for the side faces. (For example, in the case of water, it is more preferable to have a refractive index close to 1.33.) By doing so, scattering of light in the cell holding portion is further suppressed and noise is further reduced. be able to.

  As described above, when different materials are used for the bottom surface and the side surface of the cell holding portion, such a container is made of, for example, a plate of the material formed by forming a tapered hole with a refractive index. It can be produced by pasting together a plate of the above material that is larger than this and 1.5 or less. Alternatively, after molding the container with the resin of the material, the side surface of the cell holding part can be prepared by coating, bonding, and fitting with a resin having a refractive index smaller than this.

Furthermore, in the container of the present invention, the side surface of the cell holding part is preferably colored black or the like. Since the side surface is colored black or the like in this way, the reflected light in the cell holding portion can be reduced, and noise can be further reduced.
In order to color the side surface in the cell holding portion to black or the like, for example, a container may be prepared in the same manner as described above using a material obtained by kneading a black pigment or dye into the resin of the material.
Examples of the pigment used at this time include carbon black, iron oxide pigments, and composite iron oxide pigments composed of iron and other metals. Among these, carbon black is preferably used.
Moreover, it does not specifically limit as dye used at this time, Many synthetic dyes, such as an azo dye and an anthraquinone dye, are mentioned.

  On the other hand, when a fluororesin is used as the material of the container, the container often becomes opaque when the container is made of the fluororesin itself. In this case, if the bottom of the container becomes opaque, The dynamics cannot be measured accurately. Therefore, when using a fluororesin, for example, after forming a container with a transparent resin having a refractive index larger than that of the fluororesin and not more than 1.5, the fluororesin is placed on the side surface of the cell holding portion. It is preferable to use it after coating, bonding, or fitting.

In the container of the present invention, it is preferable that the side surface of the sample holding part other than the cell holding part is made of a material having a contact angle with water of 100 ° or more.
Usually, the cells to be measured are suspended in water or a buffer solution, and the liquid containing the cells is added to the sample holder. At this time, the side of the sample holder other than the cell holder is in contact with water. If the material is made of a material having an angle of 100 ° or more, the water in the liquid containing the cells is less likely to come into contact with the side surface made of the material. Cells can be moved to the cell holder.

In order to make the side surface of the sample holder other than the cell holder made of a material having a water contact angle of 100 ° or more, for example, the side contains silicon resin, fluororesin, and fluorine. The side surface portion may be formed of a material itself having a contact angle with water of 100 ° or more.
Further, for example, when MPC (2-methacryloyloxyethyl phosphorylcholine) polymer (manufactured by NOF Corporation) is coated on the side surface, protein adsorption can be reduced.

In the method for fixing cells according to the present invention, a liquid containing cells into which a labeled molecule is incorporated is added to the sample holding part of the container, and the cells are composed of vibration, pressure, gravity, centrifugal force, and electrostatic attraction. At least one selected from the above is added, and the cell is moved and fixed to the cell holding part.
Alternatively, a liquid containing cells into which a labeled molecule and magnetic particles have been incorporated is added to the sample holding portion of the container, and a magnetic force is applied to the cells so that the cells are moved to the cell holding portion and fixed. And

When applying vibration, pressure, centrifugal force, electrostatic attraction, and magnetic force to cells, each means for generating them, for example, a centrifugal machine may be used for centrifugal force, but vibration may be applied manually. . In addition, when the cells are moved to the cell holding portion by vibration, the cells may be indirectly vibrated by applying vibration to the container.
On the other hand, when the cells are moved to the cell holding part by gravity, a liquid containing cells is added to the sample holding part, and the container is allowed to stand as it is. However, in order to efficiently move the cells to the cell holding portion, it is preferable to further apply any one or more of the vibration, pressure, centrifugal force, electrostatic attractive force, and magnetic force.

When an electrostatic attraction is applied to a cell to move it to the cell holder, the outer surface of the cell is usually negatively charged, so a positive charge is generated near the cell holder, for example, under the container Then, the cell can be moved to the cell holding part by electrostatic attraction generated between these charges.
As a method for generating a positive charge in the vicinity of the cell holding unit, for example, among the electrodes connected to the power source, a negatively charged electrode is disposed on the container, and a positively charged electrode is disposed below the container. Thus, a method of sandwiching the cell holding part in the container with these electrodes can be mentioned. In this case, the time for generating a positive charge on the electrodes may be before or after the container containing the cell-containing liquid is placed between the electrodes. Alternatively, a container may be installed between the electrodes in advance, and a cell-containing solution may be added to the sample holding part before generating a positive charge on the electrode, or after generating a positive charge on the electrode, the sample holding part You may add the liquid containing a cell to.

When a magnetic force is applied to a cell to move the cell to the cell holding unit, it is necessary to incorporate fine magnetic particles into the cell in advance. A conventionally known method may be used as a method for incorporating magnetic particles into cells.
What is necessary is just to move a cell to a cell holding | maintenance part with respect to such a cell using a magnetic force generation means.

When the cell is moved to the cell holding portion by applying pressure to the cell holding portion, for example, a container in which one end of the flow path leading to the outside of the container is opened on the side surface near the bottom surface of the cell holding portion as described above. Then, a cell can be moved to the cell holding unit by connecting a suction pump to the opening at the other end of the channel and sucking the inside of the cell holding unit, that is, applying a negative pressure to the cell.
At this time, it is necessary to adjust the pressure appropriately so that the cells are not damaged. Further, it is preferable to perform suction after adding a cell-containing liquid into the sample holder.

In the present invention, a molecule whose kinetics in a cell is to be measured is labeled with a labeling compound, and then the labeled molecule is taken into a cell and its signal is measured.
A preferable example of the labeling compound used here is a fluorescent substance.
Examples of the fluorescent substance include conventionally known substances such as fluorescein, rhodamine, acriflavine, and Alexa.
A conventionally known method may be applied as a method for producing a labeled molecule and a method for incorporating a labeled molecule into a cell.

  The method for measuring intracellular molecular dynamics according to the present invention is characterized in that a signal of a labeled molecule in a cell fixed by the cell fixing method is detected from the bottom side of the cell holding part. With this measurement method, living cells can be fixed in the cell holder, and one or more cells can be fixed on the fixed cells. The tight junction can be measured.

The apparatus for measuring intracellular molecular dynamics of the present invention comprises the container for fixing cells, cell moving means, and signal detecting means.
The cell moving means is a means for applying any one of vibration, pressure, centrifugal force, electrostatic attraction, and magnetic force to the cell, and includes a vibration generating means, a pressure generating means, a centrifugal force generating means, an electric field generating means, and a magnetic force generating means. One or more selected from the group.
In the case of using the pressure generating means, the container is such that one end of the flow path leading to the outside of the container is opened on the side surface near the bottom surface of the cell holding portion as described above, and this flow path is used as the pressure transmission means. To do.

Examples of the vibration generating means include an ultrasonic generator and a mixer that stirs the liquid by vibrating a vibration plate.
Examples of the pressure generating means include a suction pump, and the pressure generating device is connected to the other end of the flow path of a container having the flow path.
Examples of the centrifugal force generating means include a tabletop centrifuge.

Moreover, as an electric field generation | occurrence | production means, the electric field generation apparatus etc. which consist of a circuit containing the two electrodes connected to the electrode can be mentioned, for example. In this case, of the two electrodes, the positively charged electrode is placed on the bottom surface side of the container and the negatively charged electrode is placed on the opening side of the container, and the container is sandwiched between these two electrodes. Thus, it is preferable to provide the electrode.
Examples of the magnetic force generating means include an electromagnet and a circuit including the electromagnet connected to a power source, or a permanent magnet. In this case, the magnetic force generating means is preferably provided on the bottom side of the container.

  As the signal detection means in the present invention, a conventionally known detection device capable of detecting signals of the various labeled molecules can be used. For example, when the labeling molecule is labeled with a fluorescent substance, a confocal fluorescence microscope or the like can be used.

In addition, the intracellular molecular dynamics measuring device of the present invention preferably has a sample addition means for adding a liquid containing cells into which a labeled molecule has been incorporated into the sample holding part of the container.
When the sample addition means is provided in the apparatus, the signal in the cells to be measured can be continuously measured in time series by automatically supplying the liquid containing the cells to the container. By doing in this way, for example, the reaction of a cell to a drug can be measured in real time, and the intracellular molecular dynamics can be measured more efficiently. Here, examples of the adding means include various dispensing devices having a function of sucking and dispensing a solution.

FIG. 1 shows a schematic configuration diagram of an example of such an apparatus for measuring intracellular molecular dynamics.
The apparatus is configured to take in a molecule labeled with a fluorescent substance into a cell and measure the fluorescence signal, and the basic apparatus configuration is based on a confocal fluorescence microscope.

The signal detection means is schematically composed of a light source 2, dichroic mirror 6, objective lens 3, bandpass filter 7, reflection prism 8, lenses 9 and 11, pinhole 10, photodetector 12, computer 13, and display 14. Has been. By irradiating light from the light source 2, the signal received by the photodetector 12 is stored in the computer 13 connected to the photodetector 12 and subjected to computation such as correlation analysis, and the result is displayed on the display 14. Is displayed.
For example, a laser generator or the like is used as the light source 2, and a weak light detector such as an avalanche photodiode (APD) or a photomultiplier tube is used as the light detector 12. The photodetector 12 is disposed with its light receiving surface aligned with the focal position of the lens 11 disposed behind the pinhole 10.

  Reference numeral 1 denotes a sample stage on which the container 4 is placed and fixed. The horizontal position and the vertical position of the container 4 are adjusted by the sample stage 1, and further, the optimum position of the cell is adjusted by finely adjusting the position of the objective lens 3 by the objective lens vertical axis adjustment mechanism (not shown). Is done.

Further, reference numeral 5 denotes a cell moving means for applying at least one selected from the group consisting of vibration, pressure, centrifugal force, electrostatic attraction, and magnetic force to the cells in the container 4. Here, an electric field generator is shown. Yes. The container 4 is moved into the electric field generator 5 by the movement of the sample stage 1 so that the cells are moved to the cell holding part of the container 4.
Reference numeral 15 denotes a sample addition means for adding a liquid containing cells into which a labeled molecule has been incorporated into the sample holding portion of the container 4, and shows a dispensing device here.

With such an apparatus for measuring intracellular molecular dynamics of the present invention, detection of intracellular signals is performed as follows.
That is, a liquid containing cells into which a labeled compound has been incorporated is added to the sample holder of the container 4 using the dispensing device 15, the sample stage 1 is moved, and the container 4 is placed in the electric field generator 5. Move. Here, after the cells are moved to the cell holding portion of the container 4, the sample stage 1 is moved, and further, the position of the objective lens 3 is finely adjusted using the objective lens vertical axis adjustment mechanism, so that the optimal cell can be obtained. The measurement position is adjusted. That is, the container is placed so that the cells are located in the confocal region of the light emitted from the light source 2 and collected by the objective lens 3.

The light emitted from the light source 2 is reflected by the dichroic mirror 6, enters the objective lens 3, and is condensed in the cell holding portion of the container 4. Since the collected light excites the fluorescent substance in the cell, fluorescence is emitted from the labeled molecule in the cell. This fluorescence again passes through the objective lens 3 and then the dichroic mirror 6 and enters the bandpass filter 7. The bandpass filter 7 allows only light in the wavelength region of the fluorescence emission spectrum, which is signal light, to pass therethrough. Thereby, scattered light generated in the container 4 and noise light such as a part of incident light reflected from the wall surface of the container 4 and returning to the incident optical path can be cut.
The signal light that has passed through the bandpass filter 7 is reflected by the reflecting prism 8 and is condensed by the lens 9 onto the pinhole surface of the pinhole 10 disposed behind the lens 9. Further, the noise light that causes the blur is removed, and the target signal light passes through the lens 11 as a signal and is detected by the photodetector 12. The computer 13 performs an operation such as correlation analysis on the signal light detected by the photodetector 12, and the result is displayed on the screen of the display 14 as an autocorrelation function or a cross-correlation function of fluorescence intensity fluctuation. Presented as a graph or data.

  Here, an apparatus suitable for measuring the signal of a fluorescent substance has been described, but the signal detection means in the apparatus may be changed as appropriate according to the type of labeling compound to be incorporated into cells and the detection method of the signal. For example, the intracellular kinetics of a molecule labeled with a labeling compound other than a fluorescent substance can be measured in the same manner.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
Example 1
Polymethylpentene (TPX (registered trademark)) was molded using a mold to produce a container 20 having a shape as shown in FIG. 2A is a schematic cross-sectional view of the container 20, and FIG. 2B is a schematic perspective view. The container 20 has a block shape with a width of 4.1 mm and a height of 2.7 mm, a flat bottom surface, and a sample holding part 21 having a truncated cone shape. Of the sample holder 21, the cell holder 22 extends from the bottom surface to a portion having a diameter of 100 μm. The opening of the sample holder 21 has a circular shape with a diameter of 3 mm, and the bottom surface of the cell holder 22 has a circular shape with a diameter of 5 μm and is flat. Moreover, the sample holding part 21 and the cell holding part 22 are molded so that the angle formed by these side surfaces 23 is a V shape with a 60 ° in the cross section. Therefore, the taper angle is 120 degrees with respect to the bottom surface of the cell holding portion.

50 μL of a cell-containing solution was added to the sample holding unit 21 of the container 20 and an attempt was made to move the cells to the cell holding unit 22 of the container 20 by the following three methods.
(1) The container 20 was set in a tabletop centrifuge (CT6D, manufactured by Hitachi Koki Co., Ltd.) and centrifuged at 1000 to 3000 rpm for 5 to 10 minutes.
(2) The container 20 was vibrated using a test tube mixer (TM-250 manufactured by IWAKI), which is a device for stirring the liquid by vibrating the diaphragm.
(3) As shown in FIG. 6, with the container 20 sandwiched vertically, the negatively charged electrode 61 is on the opening side of the container 20, and the positively charged electrode 62 is on the bottom side of the container 20. Electrostatic attractive force was applied to the cells in the container 20 by the electric field generators arranged respectively. However, these electrodes have holes 63 at positions corresponding to the positions of the cell holding portions 22 so that the cells in the container 20 can be observed and measured. Usually, since the cell outer surface is negatively charged, it is possible to efficiently attract the cells to the cell holding unit 22.
Thereafter, when the cell holding part 22 of the container 20 was observed with a microscope, one cell was held in the cell holding part 22 by any of the methods (1) to (3). It was confirmed that a plurality of cells were retained.

(Example 2)
PMMA was molded using a mold to produce a container 30 having a shape as shown in FIG. 3A is a schematic cross-sectional view of the container 30, and FIG. 3B is a schematic perspective view. The container 30 has a sample holding portion 31 having a block shape with a width of 4.1 mm and a height of 2.7 mm, a flat bottom surface, and a shape in which three truncated cones are connected. The opening is circular with a diameter of 3 mm, and the bottom is circular with a diameter of 5 μm and flat. The cell holding portion 32 extends from the bottom surface to a portion having a diameter of 30 μm, and the cross section is molded so that the angle formed by the side surface 33 is a V-shape of 60 degrees. Therefore, the taper angle is 120 degrees with respect to the bottom surface of the cell holding portion.

Except for using the container 30 having the shape shown in FIG. 3, similar to Example 1, an attempt was made to move cells to the cell holding portion 32 of the container 30 by three methods.
As a result, as in Example 1, it was confirmed that, by any method, one cell was held in the cell holding unit 32, and a plurality of cells were held on the cell.

(Example 3)
A container 40 having a shape in which one flow path as a pressure transmission means is added to the side surface near the bottom surface of the cell holding portion of a container having the same shape as that of the second embodiment is formed by resin molding as in the second embodiment Manufactured. FIG. 4 shows a schematic sectional view thereof. One end of the channel 44 is open to the side surface near the bottom surface of the cell holding portion 42, and the other end is open to the bottom surface of the container 40, and the inner diameter is 1.5 μm.
Then, a cassette tube pump (manufactured by IWAKI, PST-350, not shown) was connected to the opening on the container bottom side of this flow path.

Next, 50 μL of the cell-containing solution was added to the sample holder 41 of this container. Subsequently, the cassette tube pump is operated to depressurize the inside of the flow path 44 and the cell holding unit 42, the liquid in the sample holding unit 41 is sucked out of the container 40, and the cells are moved to the cell holding unit 42. I tried to do that.
As a result, it was confirmed that one cell was held in the cell holding unit 42 and that a plurality of cells were held on the cell.

Example 4
As shown in FIG. 5, a microplate having 96 wells (16 × 6) having a circular opening with a diameter of 6 mm was manufactured. The well 50 includes a black resin member 51 and a glass member 52. The resin member 51 is obtained by molding polystyrene containing carbon black using a mold, and the glass member 52 has a thickness of 0. .2 mm non-fluorescent soda lime glass plate. Further, the bottom surface of the resin member 51 and the bottom surface and the top surface of the glass member 52 are both flat.
The resin member 51 has a sample holding part 511 having a shape in which two truncated cones are connected at the bottom, and the opening is circular with a diameter of 50 μm. Further, a circular hole having a diameter of 5 μm is opened at a portion corresponding to the bottom surface of the cell holding portion. The cell holding portion 512 extends from the bottom surface to a portion having a diameter of 30 μm, and the cross section is molded so that the angle formed by the side surface is a V-shape with 60 degrees. Therefore, the taper angle is 120 degrees with respect to the bottom surface of the cell holding portion. The sample holders 511 have a structure in which 120 pieces are arranged in the diameter direction so that the openings are in contact with each other.
A microplate having such a well 50 was manufactured by bonding the outside of the bottom surface of the resin member 51 and the glass member 52 using an adhesive.

(Example 5)
Magnetic particles were introduced into HeLa cells, which are human cervical cancer cells, by a conventional method using a magnetic particle introduction kit Magnetfection Kit CombiMag (manufactured by OZ Bioscience) and a GFP fusion estrogen receptor DNA plasmid.
Subsequently, 50 μL of a liquid containing cells into which the magnetic particles had been introduced was added to the sample holder of the container manufactured in Examples 1 and 2. And after putting on a commercially available magnetic plate for 10 minutes, when the cell holding part of these containers was observed with the microscope, one cell was hold | maintained at the cell holding part, and also a plurality of cells were placed on the cell. Was confirmed to be retained. That is, by applying a magnetic force to the magnetic particles taken into the cells, the cells could be efficiently moved to the cell holder.

(Example 6)
The intracellular molecular dynamics were measured using the apparatus shown in FIG. 1 having an electric field generator as the cell moving means 5 and the microplate used in Example 4 as a container.
That is, by a conventional method, vector DNA in which EGFP (Enhanced Green Fluorescence Protein) is fused with an estrogen receptor (hereinafter abbreviated as ER) expression gene is introduced into HeLa cells by lipofection, and this fluorescent substance (hereinafter referred to as EGFP- A solution containing HeLa cells into which ER was abbreviated) was added to the sample addition part of the container 4. Next, the cells are moved to the cell holding part of the container 4 using the electric field generator 5 to obtain a fluorescence image of the single cell, and the fluctuation of EGFP-ER in the cell is measured by FCS, and the translational diffusion is performed. Time was calculated. The results are shown in FIG. FIG. 7A shows a fluorescence image of the EGFP-ER-expressing cell, and FIG. 7B shows a graph of the translational diffusion time of EGFP-ER in the cell.

  Subsequently, 17β-estradiol, which is a kind of estrogen, was added to the EGFP-ER-expressing HeLa cells in a cell culture solution to a final concentration of 10 nM. 17β-estradiol, which is a ligand of ER, binds to ER in the cell, and ER binds to an estrogen response element (hereinafter abbreviated as ERE) on cell DNA by forming a dimer. FIG. 8 shows a fluorescence image 30 minutes after adding 17β-estradiol to the cell culture medium. Upon addition of 17β-estradiol, EGFP-ER binds to ERE of cellular DNA. Since EGFP-ER is bound on the ERE of the nuclear matrix, the area where no EGFP-ER molecule is present as in the image was observed as black spots.

  On the other hand, the fluorescent dye Alexa647 excited by light having a wavelength of 633 nm is labeled by binding to the end of a DNA oligomer having a sequence that specifically binds to ER according to a conventional method. The HeLa cells and the EGFP-ER expressing HeLa cells were transfected respectively. These cells were moved to the cell holding part of the container 4 to obtain a fluorescence image of one cell. Further, the fluctuation of Alexa 647 in the cell was measured by FCS, and the translational diffusion time was calculated. The results are shown in FIG. 9 and FIG.

FIG. 9A shows a fluorescence image of HeLa cells transfected with Alexa647-binding DNA oligomer, and FIG. 9B shows a graph of the translational diffusion time of Alexa647 in the cells.
FIG. 10 (a) is a fluorescence image of EGFP-ER-expressing HeLa cells transfected with Alexa647-binding DNA oligomer when irradiated with light having a wavelength of 633 nm, and FIG. 10 (b) is a wavelength of 488 nm of the cells. The fluorescence image when irradiating with light is shown.
FIG. 10C is a graph of the translational diffusion time of Alexa 647 in the cell when irradiated with light having a wavelength of 633 nm, and FIG. 10D is a graph showing the intracellular diffusion time when irradiated with light having a wavelength of 488 nm. The graph of the translational diffusion time of EGFP-ER in each is shown.

From these results, when the EGFP-ER-expressing HeLa cell was transfected with Alexa647-binding DNA oligomer, the translational diffusion time was increased as shown in the graph of FIG. It was confirmed that Alexa647-binding DNA oligomer was bound to ER in EGFP-ER-expressing HeLa cells.
In addition, EGFP-ER-expressing HeLa cells transfected with Alexa647-binding DNA oligomer, both with and without 17β-estradiol, were transfected with Alexa647-binding DNA oligomer as shown in FIG. 11 (b). The translational diffusion time was longer than that of the cells, and it was confirmed that Alexa647-binding DNA oligomer was bound to ER in the EGFP-ER-expressing HeLa cells.

(Comparative Example 1)
An attempt was made to measure intracellular molecular dynamics in the same manner as in Example 6 except that a conventional microplate was used as a container. As a result, the cells in the container moved during the measurement, and the fluorescence in the cells. The signal measurement was not possible on the way.

  As described above, using the container of the present invention, it was confirmed that the measurement target cell into which the labeled molecule was incorporated could be fixed to the cell holding unit, and the intracellular molecular dynamics measuring apparatus of the present invention was used. Thus, it was confirmed that the signal of the labeled molecule was detected and the molecular dynamics in the cell could be measured easily and with high accuracy. In the above-described embodiments, examples of fluorescent labeling have been described. Detection by fluorescence is preferable because the signal intensity is strong. However, it is also possible to detect chemiluminescence or bioluminescence by applying the present invention. Chemiluminescence and bioluminescence have the advantage that detection with good signal-to-noise ratio can be performed because the signal intensity is weak but the noise is very small.

  Since molecular dynamics in living cells and tight junctions between cells can be measured easily and with high accuracy, it is useful for screening methods for drug discovery candidate substances.

It is a schematic block diagram which shows an example of the measuring apparatus of the intracellular molecular dynamics of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows an example of the container of this invention. It is the schematic which shows the example of installation of the container of this invention with respect to the cell movement means in the measuring device of the intracellular molecular dynamics of this invention. (A) of the EGFP-ER expression HeLa cell in Example 6 of this invention is a fluorescence image, (b) is a graph of the translational diffusion time of EGFP-ER. It is the fluorescence image of this cell when 17 (beta) -estradiol is added to the EGFP-ER expression HeLa cell in Example 6 of this invention. In Example 6 of the present invention, (a) of HeLa cells transfected with labeled DNA is a fluorescence image, and (b) is a graph of translational diffusion time of labeled DNA. In Example 6 of the present invention, (a) and (b) of HeLa cells labeled with EGFP-ER and labeled DNA are fluorescent images, (c) is a graph of translational diffusion time of labeled DNA, (d) is It is a graph of the translational diffusion time of EGFP-ER. It is a graph which shows the change of the translational diffusion time of EGFP-ER and labeled DNA in Example 6 of this invention.

Explanation of symbols

20 ... Container, 21 ... Sample holder, 22 ... Cell holder, 23 ... Side

Claims (16)

A container having a cell holding portion having a circular bottom surface with a diameter of 2 μm, a shape on which cells can be placed and is flat, and a side surface having a tapered shape;
The container is characterized in that the bottom surface of the container is flat, and the cell holding part is a bottom part of a sample holding part for adding and holding a liquid containing cells.   The container according to claim 1, wherein a bottom surface of the cell holding portion is a circle having a diameter of 2 μm or more.   The container according to claim 1 or 2, wherein the height of the cell holding part is 4 µm or more.   The container according to any one of claims 1 to 3, wherein the entire side surface of the sample holder is tapered.   The container according to any one of claims 1 to 4, wherein one end of a flow path leading to the outside of the container is opened on a side surface near the bottom surface of the cell holding portion.   The container according to any one of claims 1 to 5, wherein the container is made of a material having a refractive index of 1.5 or less.   The container according to any one of claims 1 to 6, wherein a side surface of the sample holding unit other than the cell holding unit is made of a material having a contact angle with water of 100 ° or more.   The container according to any one of claims 1 to 7, wherein a side surface of the cell holding part is colored black.   The container according to any one of claims 1 to 8, wherein the container has a plurality of sample holders, and the distance between the centers of the bottom surfaces of the cell holders adjacent to each other is 1 mm or more.   A liquid containing cells into which a labeled molecule has been incorporated is added to the sample holding part of the container according to any one of claims 1 to 9, and vibration, pressure, gravity, centrifugal force, electrostatic attraction is applied to the cells. A method for fixing a cell, comprising adding at least one selected from the group consisting of: moving the cell to a cell holding part, and fixing the cell.   A liquid containing cells into which a labeled molecule and magnetic particles have been incorporated is added to the sample holder of the container according to any one of claims 1 to 9, and a magnetic force is applied to the cells to hold the cells. A method for fixing cells, which is fixed by moving to a part.   The method for fixing cells according to claim 10 or 11, wherein the labeling molecule is a molecule labeled with a fluorescent substance.   A measurement of intracellular molecular dynamics, wherein a signal of a labeled molecule in a cell fixed by the cell fixing method according to any one of claims 10 to 12 is detected from the bottom side of the cell holding part. Method.   A cell moving means for adding at least one selected from the group consisting of vibration, pressure, centrifugal force, electric field, and magnetic force to the container according to any one of claims 1 to 9 and the cells in the sample holding portion of the container. And a signal detection means for detecting the signal of the labeled molecule in the cell.   The apparatus for measuring intracellular molecular dynamics according to claim 14, further comprising sample addition means for adding a liquid containing cells into which a labeled molecule has been incorporated into the sample holding part of the container. The apparatus for measuring intracellular molecular dynamics according to claim 14 or 15, wherein the labeled molecule is a molecule labeled with a fluorescent substance.

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