WO2011115189A1

WO2011115189A1 - Cell activity analysis device, cell activity analysis method and cell analysis method

Info

Publication number: WO2011115189A1
Application number: PCT/JP2011/056304
Authority: WO
Inventors: 道広秀; 雄輝柳瀬; 隆明平郡
Original assignee: 国立大学法人広島大学
Priority date: 2010-03-17
Filing date: 2011-03-16
Publication date: 2011-09-22

Abstract

The disclosed cell activity analysis device (100) is provided with: a metal thin film (5) contacting live cells (C1, C2); a prism (3) having an interface (F) substantially in contact with the metal thin film (5); a light source (1) which irradiates the prism (3) with parallel beams of polarized light (P) which then irradiate the interface (F) at a prescribed angle of incidence which induces a surface plasmon resonance phenomenon; an objective lens (6) which enlarges by a prescribed ratio an intensity image corresponding to the two-dimensional intensity distribution of the light reflected from said interface (F); an imaging unit (7) which images the enlarged intensity image; an image acquisition unit (21) which samples the image data of the intensity image; a display unit (23) and an operation unit (24) for selecting for measurement the image of at least one part of the live cells (C1, C2) from the image data of the intensity image; and an image processing unit (22) which extracts brightness values of the measurement object, and, on the basis of changes of the brightness value of the measurement object before and after application of an external stimulus to the live cells (C1, C2), calculates information relating to the change in intensity of the light reflected from the measurement object.

Description

Cell activity analyzer, cell activity analysis method, and cell analysis method

The present invention relates to a cell activity analyzer, a cell activity analysis method, and a cell analysis method for analyzing the reaction activity of living cells.

In recent years, surface plasmon resonance (SPR (surface plasmon resonance) that can measure the reaction and the amount of binding between biomolecules such as proteins and perform kinetic analysis by applying the surface plasmon resonance phenomenon and capturing the change in resonance angle in real time. resonance)) The principle of the method is used in various research and inspections.

Surface plasmon resonance consists of evanescent light that is generated when light is incident on a metal thin film under total reflection conditions, and surface plasmon that is a close-packed wave of free electrons propagating on the interface between the metal thin film and the object to be measured. It is a phenomenon that resonates. When surface plasmon resonance occurs, at least part of the energy of the incident light is transferred to surface plasmon resonance and the intensity of the totally reflected light is reduced. The incident angle at which the light intensity decreases most is called the resonance angle. This resonance angle changes according to the change in the dielectric constant of the measurement object. In the surface plasmon resonance (SPR) apparatus, the change in the dielectric constant of the measurement object can be observed by measuring the change in the resonance angle.

Examples of the object measured using the principle of the surface plasmon resonance method include enzymes, antibodies, DNA, cells, and the like. Among these, a method has been proposed in which a cell is an object to be measured, and an external stimulation activity for a living cell is evaluated using a surface plasmon resonance apparatus (see Patent Document 1). In this method, a surface plasmon resonance apparatus is used to evaluate the activity of external stimuli on live cells using as an index the secondary signal that appears after the primary signal observed when live cells are exposed to external stimuli. Yes. The signal is a dielectric constant, refractive index, or resonance angle measured in the same manner as a normal SPR measurement for living cells (hereinafter, appropriately described by any one of dielectric constant, refractive index, and resonance angle). Indicates a change.

Furthermore, as a measurement object of cells, Patent Document 2 describes a method and system for simply calculating the number contained in a cell population fixed on a plate. For example, the method is a method for analyzing a cell, the step of measuring reflected light intensity caused by a cell fixed on a plate using surface plasmon resonance imaging, and the reflected light intensity from the cell A step of calculating a parameter relating to. Specifically, a method for analyzing a cell-related parameter such as the number of cells, cell adhesion area, or cell size is described.

In addition to this, Non-Patent Document 1 describes a method for analyzing parameters relating to cells such as cell adhesion density using a surface plasmon resonance apparatus.

Japanese Patent No. 3795112 JP 2004-271337 A

However, although the method described in Patent Document 1 can evaluate the average value of the activity of external stimuli on a plurality of living cells in real time, it analyzes and evaluates the activity of external stimuli on individual living cells. It is difficult.

In addition, as described above, the method and apparatus for analyzing cells described in Patent Document 2 and the method described in Non-Patent Document 1 are parameters related to cells such as the number of cells, the adhesion area of cells, or the size of cells. It is the method and apparatus (system) aiming at calculation and analysis of. Therefore, when such a method and apparatus (system) is used, it is impossible to directly analyze and evaluate stimulus responses including parameters other than those described above for living cells. Difficult to do.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell activity analyzer, a cell activity analysis method, and a cell analysis method capable of analyzing the activity of external stimuli for individual living cells. And

In order to achieve the above object, a cell activity analyzer according to the first aspect of the present invention comprises:
A cell activity analyzer that analyzes the activity of external stimuli on living cells using the surface plasmon resonance phenomenon,
A metal thin film in contact with the living cell on one side;
A refractive optical element having an interface substantially in contact with the other surface of the metal thin film;
Incident means for causing a P-polarized parallel light beam to enter the refractive optical element and to enter the interface at a predetermined incident angle that causes the surface plasmon resonance phenomenon;
A magnifying optical system for enlarging an intensity image corresponding to a two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface to a predetermined magnification;
Imaging means for capturing the intensity image magnified by the magnification optical system;
Image acquisition means for sampling image data of the intensity image captured by the imaging means;
Selecting means for selecting at least a partial image of the living cells as a measurement target from the image data of the intensity image sampled by the image acquisition means;
The brightness value of the measurement target selected by the selection means is extracted, and the reflected light of the measurement target is based on a change in the brightness value of the measurement target before and after the external stimulus is applied to the living cells. A calculating means for calculating information on a change in intensity of
Is provided.

Preferably, the selection means can specify a plurality of measurement objects,
The calculation means extracts a luminance value of each of the plurality of selected measurement objects from the image data of the intensity image, and calculates information regarding a change in intensity of reflected light of the measurement object for each measurement object. .

More preferably, the selection means selects a plurality of different locations in the same live cell image as the measurement target.

Preferably, the calculation means is based on a difference between a luminance value of the measurement target before applying the external stimulus to the living cells and a luminance value of the measurement target after applying the external stimulus. Then, information on the change in the intensity of the reflected light of the measurement target is calculated.

Preferably, the calculation unit corrects information related to a change in intensity of reflected light of the measurement target based on a luminance value component of a portion where the living cells do not exist in the image data of the intensity image.

More preferably, the predetermined incident angle is equal to a resonance angle when the living cell is not in contact with the metal thin film.

More preferably, it further comprises analysis means for analyzing the living cells based on information on a change in intensity of reflected light of the measurement target calculated by the calculation means.

More preferably, the analysis means extracts a characteristic of a change pattern with time of the dielectric constant of the living cell to be measured.

More preferably, the analysis means determines whether the time-dependent change pattern of the dielectric constant of the living cell corresponds to a monophasic, biphasic, triphasic or other atypical pattern.

Also preferably, a microscope is further provided for observing the living cells in contact with the metal thin film from the one surface side.

Preferably, the selection means includes
Display means for displaying an image based on the image data of the intensity image sampled by the image acquisition means;
An operation means for designating at least a partial image of the living cell as the selected measurement object from image data of the intensity image sampled by the image acquisition means by an operation input;
Is further provided.

Preferably, a plurality of the thin metal films can be arranged by separating a group of living cells including the living cells.

More preferably, it further includes external stimulus applying means for applying different external stimuli to each of the plurality of living cell groups.

The cell activity analysis method according to the second aspect of the present invention comprises:
A cell activity analysis method for analyzing the activity of external stimuli on living cells using surface plasmon resonance phenomenon,
An arrangement step of arranging the living cells so as to contact one surface of the metal thin film;
An incident step in which a P-polarized parallel light beam is incident on a refractive optical element having an interface substantially in contact with the other surface of the metal thin film, and is incident on the interface at a predetermined incident angle that causes the surface plasmon resonance phenomenon; ,
An enlargement step of enlarging an intensity image corresponding to a two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface to a predetermined magnification by an enlargement optical system;
An imaging step of capturing the intensity image magnified by the magnification optical system;
An image acquisition step of sampling image data of the intensity image imaged in the imaging step;
From the image data of the intensity image sampled by the image acquisition step, a selection step of selecting at least a partial image of the living cell as a measurement target;
The brightness value of the measurement target selected by the selection step is extracted, and the reflected light of the measurement target is based on a change in the brightness value of the measurement target before and after the external stimulus is applied to the living cells. A calculation step for calculating information on a change in intensity of the
Including
The activity of the external stimulus with respect to at least a part of the living cells is analyzed using information on the change in the intensity of reflected light of the measurement target calculated in the calculation step as an index.

Preferably, in the selection step, a plurality of measurement objects can be specified.
In the calculation step, a luminance value of each of the plurality of selected measurement objects is extracted from the image data of the intensity image, and information regarding a change in intensity of reflected light of the measurement object is calculated for each measurement object. .

More preferably, in the selection step, a plurality of different locations in the same live cell image are selected as the measurement target.

Preferably, in the calculation step, based on a difference between a luminance value of the measurement target before applying the external stimulus to the living cells and a luminance value of the measurement target after applying the external stimulus. Then, information on the change in the intensity of the reflected light of the measurement target is calculated.

Preferably, in the calculation step, information on a change in intensity of reflected light of the measurement target is corrected based on a luminance value component of a portion where the living cells do not exist in the image data of the intensity image.

Also preferably, the predetermined incident angle is equal to a resonance angle when the living cell is not in contact with the metal thin film.

Most preferably, the selection step comprises
A display step of displaying an image based on the image data of the intensity image sampled in the image acquisition step;
By an operation input, an operation step of designating at least a part of the live cell image as the selected measurement object from the image data of the intensity image sampled in the image acquisition step;
Further included.

Preferably, in the arrangement step, a plurality of living cell groups including the living cells are arranged apart from each other.

More preferably, a different external stimulus is applied to each of the plurality of living cell groups.

The cell analysis method according to the third aspect of the present invention comprises:
The living cells are analyzed based on information relating to changes in the intensity of reflected light of the living cells obtained by analysis using the cell activity analysis method of the present invention.

Preferably, the living cells are analyzed by extracting characteristics of a change pattern of the dielectric constant of the living cells over time based on information on a change in intensity of reflected light of the measurement target.

More preferably, it is determined whether the time-dependent change pattern of the dielectric constant of the living cells corresponds to a monophasic, biphasic, triphasic or other atypical pattern.

More preferably, cancer cells are the subject of analysis.

More preferably, the cancer cell is any of a stomach cancer cell, a prostate cancer cell, or an angiosarcoma cell.

More preferably, the living cells exposed to the external stimulus by cytokines are analyzed.

More preferably, the living cells exposed to the external stimulus by EGF are analyzed.

According to the present invention, the image data of the reflection intensity image of the P-polarized parallel light beam incident on the interface substantially in contact with the metal thin film with which the living cells are in contact is acquired by sampling. Then, from the acquired image data, at least a part of the image of the living cell is selected as the measurement target, and information on the change in the intensity of the reflected light of the measurement target is calculated based on the change in the luminance of the selected image. . A living cell to be measured is a dielectric, and its dielectric constant changes depending on a response to an external stimulus. As a result, the resonance angle of the surface plasmon resonance phenomenon changes, and the intensity of the reflected reflected light changes there. Therefore, if the information on the calculated change in the intensity of the reflected light is calculated, the activity of the external stimulus for each living cell related to the reflected light can be analyzed based on the information. According to the present invention, it is possible to evaluate and analyze the properties of individual living cells and / or a part of each living cell, or to isolate specific types of living cells. It becomes possible to detect that the reaction is different, and it becomes possible to analyze individual cells without isolation, and to analyze each individual place in each individual cell. As a result, it is possible to detect the behavior of activity for each individual living cell.

It is a schematic diagram which shows the structure of the cell activity analyzer which concerns on embodiment of this invention. It is a graph which shows an example of the incident angle dependence of the intensity | strength of the reflected light in the cell activity analyzer of FIG. FIGS. 3A to 3H are diagrams illustrating an example of an image of a reflection intensity image captured when the incident angle is changed by 1 °. FIGS. 4A to 4C are diagrams illustrating an example of temporal changes in the image of the reflection intensity image when the living cells (RBL-2H3 cells) are not stimulated. FIG. 5A to FIG. 5C are diagrams showing an example of temporal changes in the image of the reflection intensity image when a living cell (RBL-2H3 cell) is stimulated. FIG. 6A is a graph illustrating an example of a temporal change in the intensity of reflected light of a measurement target that is not stimulated. FIG. 6B is a graph showing an example of a temporal change in the intensity of reflected light of a measurement target stimulated with living cells (RBL-2H3 cells). It is a block diagram which shows the detailed structure of the image processing part of FIG. It is a figure for demonstrating the various components contained in the intensity | strength of reflected light. FIGS. 9A to 9C are diagrams showing an example of changes in the intensity of reflected light in different parts of a living cell (RBL-2H3 cell). It is a flowchart of an example of a cell activity analysis method (analyte subject cell analysis). It is a figure which shows the other example of a metal thin film. FIG. 12A is a perspective view of a part of a cell activity analyzer equipped with a multiwell chamber, and FIG. 12B shows an image captured by the cell activity analyzer of FIG. It is a figure which shows an example. 13A is a diagram showing an example of an image of a reflection intensity image when the PAM212 cell is stimulated and FIG. 13B is a diagram when the A431 cell is stimulated. FIG. 14A is a graph showing an example of temporal change in intensity of reflected light when PAM212 cells are stimulated and FIG. 14B is stimulated with A431 cells. It is a figure which shows the sensor chip | tip with which the RBL-2H3 cell couple | bonded with the anti- DNP-mouse IgE and the RBL-2H3 cell which has not couple | bonded are arrange | positioned. 16 is a graph showing an example of a temporal change in intensity of reflected light when stimulated with DNP-HSA and PMA in the sensor chip shown in FIG. FIGS. 17A to 17C are diagrams illustrating an example of a temporal change in the image of the reflection intensity image when the sensor chip illustrated in FIG. 15 is stimulated with DNP-HSA and PMA. FIG. 4 is a diagram showing a sensor chip on which RBL-2H3 cells bound with anti-DNP-mouse IgE and RBL-3D4 cells bound with a human IgE antibody are arranged. FIG. 19 is a graph showing an example of a temporal change in intensity of reflected light when stimulated with DNP-HSA and an anti-human IgE antibody in the sensor chip shown in FIG. FIGS. 20A to 20C are diagrams illustrating an example of a temporal change in the image of the reflection intensity image when the sensor chip illustrated in FIG. 18 is stimulated with DNP-HSA and anti-human IgE. It is a figure which shows the sensor chip | tip with which RBL-2H3 cell and A431 cell which couple | bond anti-DNP-mouse IgE are arrange | positioned. FIGS. 22A and 22B are diagrams showing an example of the temporal change in reflection intensity when the sensor chip shown in FIG. 21 is stimulated with DNP-HSA and EGF. FIG. 23A and FIG. 23B are diagrams illustrating an example of a temporal change in an image of a reflection intensity image when stimulated with DNP-HSA and EGF in the sensor chip illustrated in FIG. It is a figure which shows the change of the resonance angle with time by EGF stimulation in the CHO cell which forcedly expressed wild type human EGFR. It is a figure which shows the result of the western blotting by the anti- phosphorylation specific EGFR antibody, the anti- EGFR antibody, and the anti- FLAG antibody in the CHO cell which expressed the human EGFR with which the ATP binding domain which concerns on the Example was mutated. It is a figure which shows the measurement result of the expression level of EGFR in the CHO cell surface in which the human EGFR with which the ATP binding domain which concerns on an Example was expressed was expressed. It is a figure which shows the change of the resonance angle with time by EGF stimulation in the CHO cell which expressed the human EGFR with which the ATP binding domain was mutated. It is a figure which shows the change of the resonance angle with time by EGF stimulation in the gastric cancer cell line MKN-1. It is a figure which shows the change of the resonance angle with time by EGF stimulation in the gastric cancer cell line MKN-7. It is a figure which shows the change of the resonance angle with time by EGF stimulation in the gastric cancer cell line MK28. It is a figure which shows the time-dependent change of the resonance angle by EGF stimulation in the prostate cancer cell line DU145. It is a figure which shows the time-dependent change of the resonance angle by EGF stimulation in the prostate cancer cell line LNCap. FIG. 33 (A) is a diagram showing the results of Western blotting using an anti-phosphorylation-specific EGFR antibody and an anti-EGFR antibody of a human hemangiosarcoma cell line. FIG. 33 (B) is a graph showing changes in resonance angle over time by EGF stimulation in a human hemangiosarcoma cell line.

Embodiments of the present invention will be described in detail with reference to the drawings.

The cell activity analyzer 100 according to the embodiment of the present invention is an apparatus that analyzes the activity observed when a living cell as a subject is exposed to an external stimulus using a surface plasmon resonance phenomenon. The temperature of the space in which the cell activity analyzer 100 is installed is preferably adjusted to 37 ° C. by a thermostat (not shown), but is not limited thereto.

First, the configuration of a cell activity analyzer 100 according to the present embodiment will be described with reference to FIG. As shown in FIG. 1, the cell activity analyzer 100 includes a light source 1, a polarizing plate 2, a prism 3, a glass substrate 4, a metal thin film 5, an objective lens 6, an imaging unit 7, and a computer 8. , A flow cell 9, a liquid supply unit 10, and a microscope 11. In the present embodiment, the optical axis of the optical system including the light source 1, the polarizing plate 2, the prism 3, the glass substrate 4, the metal thin film 5, the objective lens 6, and the imaging unit 7 is defined as AX. .

The light source 1 is, for example, a semiconductor laser. This semiconductor laser oscillates and outputs laser light having a wavelength of 635 nm, for example. The laser beam output from the light source 1 is converted into a parallel light beam by a collimator lens (not shown) or the like and enters the polarizing plate 2. In addition, as the light source 1, you may use red, white LED (Light * Emitting * Diode), etc. FIG.

The polarizing plate 2 converts the incident laser light into a linearly polarized parallel light beam and emits it. This linearly polarized light becomes P polarized light with respect to an interface F between a glass substrate 4 and a metal thin film 5 described later. The light source 1 and the polarizing plate 2 correspond to the incident means.

As the prism 3, for example, S-LAL-10 glass is employed. The refractive index of this glass is 1.72. The prism 3 receives the parallel light flux that has been changed to P-polarized light by the polarizing plate 2.

For the glass substrate 4, for example, S-LAL-10 glass is adopted. That is, the glass substrate 4 and the prism 3 have the same refractive index. Both are bonded by a matching oil having a refractive index of 1.72. Thereby, the laser light (P-polarized light) incident on the prism 3 is incident on the glass substrate 4 and goes straight as it is. The prism 3 and the glass substrate 4 correspond to a refractive optical element.

A metal thin film 5 is deposited on the glass substrate 4. The metal thin film 5 is, for example, a gold film. In addition, thin films such as Ag, Cu, Zn, Al, and K can also be used as the metal thin film 5. The thickness of the metal thin film 5 is, for example, 50 nm. The metal thin film 5 is formed on the glass substrate 4 by vapor deposition, for example. The laser light incident on the glass substrate 4 is incident on the interface F between the glass substrate 4 and the metal thin film 5 at an incident angle θ = 56 ° that satisfies the total reflection condition and generates the surface plasmon resonance phenomenon. If the metal thin film 5 is not installed, the laser light is totally reflected at the interface F. *

The laser light reflected by the interface F is emitted from the glass substrate 4 and the prism 3 and enters the objective lens 6. The objective lens 6 refracts and emits laser light. In the objective lens 6, a lens having a longer rear focal length than a front focal length is used. Therefore, the objective lens 6 enlarges the object image at a predetermined magnification and forms an image on the image plane. The laser light emitted from the objective lens 6 reaches the imaging surface of the imaging unit 7.

The imaging unit 7 is, for example, a CCD (Charge-Coupled Device) image sensor or a CMOS (Complementary Metal-Oxide Semiconductor) image sensor. The imaging unit 7 receives the laser light reflected by the interface F. The imaging surface of the imaging unit 7 and the interface F are in a conjugate relationship. Therefore, an intensity image corresponding to the two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface F of the prism 3, that is, a reflection intensity image is formed on the imaging surface of the imaging unit 7.

This reflection intensity image is magnified by, for example, 2 to 40 times by the objective lens 6. The imaging unit 7 captures the reflection intensity image. The imaging unit 7 outputs an image signal corresponding to the reflected intensity image.

The image signal output from the imaging unit 7 is input to the computer 8. The computer 8 has a CPU and a memory (both not shown). When the CPU executes the program stored in the memory, the functions of the image acquisition unit 21, the image processing unit 22, the display unit 23, and the operation unit 24 illustrated in FIG. 1 are realized.

The image acquisition unit 21 samples the input image signal at regular time intervals, and outputs the image data obtained by the sampling to the image processing unit 22. The image processing unit 22 outputs the image data acquired by the image acquisition unit 21 to the display unit 23. The display unit 23 displays an image based on the input image data.

The operation unit 24 is a user interface that receives user operation input, and includes, for example, a keyboard, a touch panel, and a mouse. The operation unit 24 is operated by a user who views an image displayed on the display unit 23. The user operates the operation unit 24 to specify a specific measurement target in the image of the reflection intensity image displayed on the display unit 23, for example. By the operation input of the operation unit 24, the position coordinates of the measurement target in the designated image are input to the image processing unit 22.

The image processing unit 22 determines the luminance value of the measurement target specified by the operation input of the operation unit 24 from the sampled reflection intensity image (if the measurement target is a region, the average value of the luminance values of that region). , And information on the temporal change in the intensity of the reflected light of the measurement target is calculated based on the extracted temporal change in the luminance value of the measurement target. This time change in the intensity of the reflected light corresponds to a time change in the dielectric constant of the measurement target. The image processing unit 22 graphs the information related to the temporal change in the calculated reflected light intensity of the measurement target, and outputs the image data of the graph to the display unit 23.

The display unit 23 displays simultaneously the image of the reflection intensity image obtained by sampling and the image of the graph showing the temporal change in the intensity of the reflected light in the designated measurement target. The user can analyze the activity of the external stimulus with respect to the living cells while referring to the graph showing the temporal change of the intensity of the reflected light.

On the surface (one surface) opposite to the interface F in the metal thin film 5, live cells C1 and C2, which are measurement targets of the active reaction, are attached. The method of attaching the living cells C1 and C2 to the metal thin film 5 is performed by using, for example, an appropriate spacer (poly-L-lysine or the like) between the living cells C1 and C2 and the metal thin film 5, and living cells C1 and C2. Although any method can be used as long as it is known in the art, any method may be used. Furthermore, a method of utilizing the affinity of the cell membrane for lipid, a method of covalently tethering, or a method of tethering with a positive charge developed by the present inventors (Yanase et al. 2007. Biosensors Bioelectron. 23, 562-567 and JP 2007-14327 A).

On the metal thin film 5, a flow cell 9 is provided as a flow path for flowing a liquid. The flow cell 9 is a flow path for flowing a liquid to be exposed to the living cells C1 and C2 set on the metal thin film 5. The flow cell 9 is connected to the liquid supply unit 10. A liquid to be exposed to the living cells C1 and C2 is supplied from the liquid supply unit 10 into the flow cell 9.

The liquid flowing through the flow cell 9 contains, for example, an antigen that may bind to the antibodies on the living cells C1 and C2. When the antigen binds to the antibody on the living cells C1 and C2, the living cells C1 and C2 are stimulated and activated (see, for example, Patent Document 1). That is, since the external stimulation is applied to the living cells C1 and C2 by exposing the living cells C1 and C2 to the liquid by the flow cell 9 and the liquid supply unit 10, in this embodiment, the flow cell 9 and the liquid supply unit 10 are stimulated. Corresponds to the granting means. In other words, the flow cell 9 and the liquid supply unit 10 correspond to an exposure unit that exposes a living cell as a subject to be exposed to an external stimulus. The exposure means holds the living cells to be examined as a subject in an environment of a predetermined condition, and exposes the living cells of the subject to a given external stimulus.

The microscope 11 is installed to observe the living cells C1 and C2 set on the metal thin film 5 from the opposite side of the interface F. The metal thin film 5 is provided with alignment marks (not shown) in advance. This mark is provided at a position that falls within both the imaging field of the microscope 11 and the imaging field of the imaging unit 7. With this mark, the positional relationship between a specific portion in the observation image of the microscope 11 and a specific portion of the image captured by the imaging unit 7 becomes clear. With reference to the image of the mark appearing in both images, the position of the specific living cells C1 and C2 that appear in the image of the reflection intensity image captured by the imaging unit 7 within the observation field of the microscope 11 is specified, for example, pipetting operation, etc. In addition, the living cells C1 and C2 can be taken out using a method known in the art.

The graph of FIG. 2 shows an example of the incident angle dependence of the intensity of the reflected light received by the imaging unit 7. In this graph, the horizontal axis indicates the incident angle θ of the parallel light flux to the interface F, and the vertical axis indicates the intensity of the reflected light at the incident angle θ. FIG. 2 shows three characteristic curves (a) to (c).

The characteristic curve (a) is a curve showing the incident angle dependence of the intensity of the reflected light in a state where nothing is placed on the metal thin film 5 (the state where there are no living cells C1 and C2). According to this, the intensity of reflected light is most attenuated at an incident angle of 56 ° due to the surface plasmon resonance phenomenon. This incident angle of 56 ° is called a resonance angle. In the present embodiment, the incident angle of the laser beam to the interface F is set to 56 ° so that the intensity of the reflected light when the living cells C1 and C2 are not in contact is the darkest.

The characteristic curve (b) is a curve showing the incident angle dependence of the reflected light intensity when the living cells C1 and C2 are set on the metal thin film 5 and the living cells C1 and C2 are not yet stimulated. Since the living cells C1 and C2 are dielectrics, the dielectric constant changes around the metal thin film 5 where the living cells C1 and C2 come into contact, and the incident angle dependence of the reflected light intensity is characteristic from the characteristic curve (a). The curve (b) is shifted and the resonance angle is also shifted from 56 ° to θ1.

The characteristic curve (c) shows that the living cells C1 and C2 are set on the metal thin film 5, and the living cells C1 and C2 are stimulated by the antigen contained in the liquid flowing through the flow cell 9, and in response to the stimulation. It is a curve which shows the incident angle dependence of reflected light intensity. When the living cells C1 and C2 are stimulated and respond to the stimulation, the dielectric constants of the living cells C1 and C2 are further changed. Therefore, the incident angle dependence of the intensity of the reflected light is changed from the characteristic curve (b) to the characteristic curve (c). The resonance angle is also shifted from θ1 to θ2.

In this embodiment, the incident angle of the parallel light flux on the interface F is fixed at 56 °. Therefore, focusing attention on an incident angle of 56 °, the intensity of the reflected light is I1 and is the darkest when the living cells C1 and C2 are not in contact with the metal thin film 5. Further, when the living cells C1 and C2 adhere to the metal thin film 5, the intensity of the reflected light becomes I2, which is higher by ΔI1 than I1. Further, when the living cells C1 and C2 attached to the metal thin film 5 are stimulated by an antigen or the like and respond to the stimulation, the intensity of the reflected light increases from I2 by ΔI2 to I3, and becomes stronger.

Thus, in the image of the reflection intensity image picked up by the imaging unit 7, the place where the living cells C1 and C2 do not exist becomes dark, the place where the cells exist becomes bright, and the living cells C1 and C2 become bright. The activated area becomes brighter.

FIGS. 3A to 3H show images of reflection intensity images when the incident angle θ of the laser beam is changed by 1 ° from 53 ° to 60 °. As shown in FIGS. 3A to 3H, the contrast between the place where the live cells C1 and C2 are present and the place where the live cells C1 and C2 are not present is the highest in the incident angle. θ = 56 °. This is apparent from the fact that in FIG. 2, the resonance angle is 56 ° when the living cells C1 and C2 are not present, and the reflected light is most attenuated. That is, if the incident angle is the same as the resonance angle θ = 56 °, Δl1 can be maximized, and the contrast of the image is increased.

FIGS. 4A to 4C show changes in the image of the reflection intensity image after 0 minutes, 10 minutes, and 20 minutes in a state where the living cells C1 and C2 are not stimulated. . 5A to 5C show changes in the reflection intensity image after 0 minutes, 10 minutes, and 20 minutes after stimulating the living cells C1 and C2. . 4A to 4C and FIGS. 5A to 5C, it is clear that when the living cells C1 and C2 react to an external stimulus, the living cells C1 and C2 It can be seen that the luminance of the portion corresponding to is changing.

While viewing the reflection intensity image displayed on the display unit 23 of the computer 8, the user operates the operation unit 24 (for example, a mouse) to specify a specific measurement target in the image of the reflection intensity image displayed on the display unit 23. Can be specified. This measurement target may be specified as a point (for example, a part of one living cell) or may be specified as a region (for example, an entire region of one living cell).

The user can designate a plurality of living cells at a time by operating the operation unit 24 and selecting, for example, several bright portions shown in FIG. The image processing unit 22 creates an image of a graph of the change in the intensity of the reflected light of the portion corresponding to the designated living cell and causes the display unit 23 to display the image.

6 (A) and 6 (B) show the temporal change in the intensity of the reflected light of the portion corresponding to each of several living cells specified in this way and the time of the average value of the intensity of the reflected light. Changes are shown. In FIG. 6 (A), the time change etc. of the intensity | strength of the reflected light of each part in the state set to the metal thin film 5 without a living cell being stimulated are shown, and a living cell is stimulated in FIG. 6 (B). The time change of the intensity of the reflected light of each part in the reacted state is shown.

As can be seen by comparing FIG. 6 (A) and FIG. 6 (B), when live cells are stimulated, reacted, and activated, the intensity of the reflected light at that portion changes greatly. Note that the horizontal line at the bottom of the graph in FIG. 6B indicates that at that time, a solution (a solution containing DNP-HSA) is caused to flow through the flow cell 9 and the living cells are stimulated. 4A to FIG. 4C, FIG. 5A to FIG. 5C, FIG. 6A, and FIG. 6B will be described in detail in the embodiments described later. Explained.

By the way, the computer 8 is provided with a function for obtaining the change in the intensity of the reflected light at the designated measurement object with higher accuracy. FIG. 7 shows a further detailed configuration of the image processing unit 22 of the computer 8.

As illustrated in FIG. 7, the image processing unit 22 includes an operation content analysis unit 30, a measurement target extraction unit 31, an initial value holding unit 32, a dark component extraction unit 33,

difference units

34 and 35, and waveform generation. Part 36.

The operation content analysis unit 30 analyzes the operation content input from the operation unit 24. The operation content obtained as a result of analysis includes the position coordinates in the specified measurement target image, the position coordinates in the image at the position specified as a part where no living cells exist, the measurement start command of waveform data, etc. include.

The operation content analysis unit 30 outputs the position coordinates in the designated measurement target image to the measurement target extraction unit 31. Further, the operation content analysis unit 30 outputs a waveform data measurement start command to the measurement target extraction unit 31, the initial value holding unit 32, and the dark component extraction unit 33. Further, the operation content analysis unit 30 outputs the position coordinates in the image at the position designated as a part where no living cells exist to the dark component extraction unit 33.

When a measurement start command for waveform data is input, the measurement target extraction unit 31 extracts the luminance value of the measurement target for each measurement target based on the position coordinates of the measurement target input from the operation content analysis unit 30. Output. Note that only the luminance value of the first measurement target at the time when the waveform data measurement start command is input is output to the initial value holding unit 32.

The initial value holding unit 32 holds and outputs the luminance value of the measurement target output from the measurement target extraction unit 31 for each measurement target when the waveform data measurement start command is input. This output is held as an initial value during measurement and continues to be output.

When a waveform data measurement start command is input, the dark component extraction unit 33 extracts and outputs the amount of change in the luminance value of the portion where there is no live cell input from the operation content analysis unit 30. The initial value of this change amount is zero. After that, the luminance value obtained at the next sampling time is calculated as the amount of change.

The difference unit 34 subtracts the initial value of the measurement target luminance value output from the initial value holding unit 32 from the measurement target luminance value output from the measurement target extraction unit 31 and outputs the result.

The difference unit 35 subtracts the luminance value output from the dark component extraction unit 33 from the luminance value output from the difference unit 34 and outputs the result.

The waveform generation unit 36 converts the luminance value output from the difference unit 35 into the intensity of the reflected light, and arranges the intensity of the reflected light obtained so far in time series, thereby changing the intensity of the reflected light over time. Generate and output graph image data.

As shown in FIG. 8, the reflected light intensity I in the reflection intensity image includes (A) a change due to the reaction of the living cell and (B) a default intensity component of the living cell before giving the stimulus. And (C) a component of intensity change in a region where no living cells exist. In the image processing unit 22, the luminance value output from the measurement target extraction unit 31 corresponds to the intensity I of the reflected light, the luminance value output from the initial value holding unit 32 corresponds to the component (B), and the dark component The luminance value output from the extraction unit 33 corresponds to the component (C).

In this measurement, the components (B) and (C) consist of background and noise, respectively. Therefore, the

difference units

34 and 35 subtract the luminance value output from the initial value holding unit 32 and the luminance value output from the dark component extraction unit 33 from the luminance value output from the measurement target extraction unit 31. If the luminance value is corrected, the waveform data of the component (A) to be originally measured can be acquired with high accuracy.

Next, a method for analyzing the activity of a living cell using the cell activity analyzer 100 according to this embodiment will be described in order.

First, live cells C 1 and C 2 are set on the metal thin film 5. In this state, emission of laser light is started, a reflection intensity image is captured by the imaging unit 7, and an image of the reflection intensity image is displayed on the display unit 23. In the displayed image, the portion where the living cells C1 and C2 are present is displayed brightly. Therefore, the user who has viewed the image displayed on the display unit 23 operates the operation unit 24 to display the image in the image. The living cells C1 and C2 are designated as measurement targets, and the portion where no living cells exist is designated.

When the operation unit 24 designates a measurement target or a portion where no living cell exists, and a measurement start command is input, display of a graph of the temporal change in the intensity of reflected light on the measurement target is started. In this state, supply of a liquid containing an antigen or the like from the liquid supply unit 10 is started, and the liquid flows through the flow cell 9. As a result, the living cells C1 and C2 set on the metal thin film 5 are stimulated, and when the conditions are satisfied, the cells react with the stimulation and are activated. This step is an exposure step in which live cells to be examined are exposed to an external stimulus. The exposure method may be a method suitable for each external stimulus. For example, in the case of giving stimulation with EGF, the target living cells may be infiltrated into an EGF solution having an appropriate concentration to give the stimulation.

When the living cells C1 and C2 react, the dielectric constant changes, the reflected light intensity of the measurement object changes, and the change is measured by the cell activity analyzer 100 and appears in the graph displayed on the display unit 23. It becomes like this. This step is a dielectric constant measurement step for measuring a change with time in the dielectric constant of the cells C1 and C2 exposed to the external stimulus in the exposure step. In the dielectric constant measurement step, the measurement result of the dielectric constant of the living cell is stored together with the measured time. The time information to be measured may be a relative time from the start of measurement. The measured dielectric constant and the information on the time constitute a time-dependent change pattern of the dielectric constant of the living cells of the subject. The user analyzes the activity of the external stimulus for the living cells C1 and C2 based on the change pattern of the dielectric constant with time, that is, the time change. For example, it is possible to detect that the living cell C1 is activated but the living cell C2 is not activated, and the like that the reaction is different for each living cell.

Further, by this analysis method, when the characteristics of the temporal change in the intensity of reflected light in a specific living cell are known in advance, it is also possible to analyze the types of the living cells C1 and C2. For example, it is assumed that the waveform generation unit 36 of the image processing unit 22 stores waveform data indicating the temporal change characteristics of reflected light intensity in several living cells as analysis means. Then, the waveform generator 36 obtains the correlation between the waveform of the living cell to be measured currently being created and the waveform data already stored, and analyzes the cell having the maximum correlation as the living cell being measured. can do. On the other hand, in a state where a plurality of living cells having different shapes are mixed, when it is desired to examine the activity of external stimulation on various living cells, each of the living cells displayed on the display unit 23 is measured. If it designates as a target, it can analyze without isolating every living cell of each shape.

As described above, in the present embodiment, if individual living cells are designated as measurement targets, activation of the individual living cells due to external stimulation can be analyzed. In the case of further using the living cells that are the measurement target, the user can collect the living cells in a live state by pipetting operation or the like while observing the living cells with the microscope 11. The cell activity analyzer 100 may be further provided with a pipette device (not shown) that automatically collects live cells, and the live cells may be automatically collected using the pipette device.

Moreover, according to the cell activity analyzer 100 according to the present embodiment, as shown in FIG. 9A, a plurality of different portions of the same living cell can be designated as a measurement target. In the image shown in FIG. 9A, the measurement points P1 to P13, which are a plurality of measurement objects randomly extracted, are designated within one living cell by the operation input of the operation unit 24. Among these measurement points, P1 to P5 are measurement points near the center of the living cell, that is, near the nucleus, and P6 to P12 are measurement points near the edge of the living cell, that is, near the cell membrane. P13 is a measurement point where there is no cell.

Here, a case where a solution containing an antigen is supplied from the liquid supply unit 10 to the flow cell 9 will be described. After the liquid is supplied, the image of the reflection intensity image changes from the image shown in FIG. 9A to the image shown in FIG. 9B. FIG. 9C shows the change in the average value of the intensity of reflected light at the measurement points P1 to P5, that is, the measurement points near the center of the living cell, and the average value of the intensity of reflected light at the measurement points P6 to P12. Changes. As shown in FIG. 9C, the change in reflected light intensity is larger at the measurement point near the center of the living cell, that is, near the nucleus than at the measurement point near the edge of the living cell, that is, near the cell membrane. I can see that As a result, it becomes clear that in the stimulation with this liquid, the change in the dielectric constant in the vicinity of the nucleus is larger than in the vicinity of the cell membrane.

As described above in detail, according to the present embodiment, an image of the reflection intensity image of the P-polarized parallel light beam incident on the interface F substantially in contact with the metal thin film 5 in contact with the living cells C1 and C2 is obtained by sampling. Is done. Then, from the acquired image data, at least a part of the images of the living cells C1 and C2 is selected as the measurement target, and information on the change in the intensity of the reflected light of the measurement target is obtained based on the change in the luminance of the selected image. Calculated.

The living cells C1 and C2 to be measured are dielectrics, and the dielectric constant changes due to a reaction caused by an external stimulus. When the dielectric constants of the living cells C1 and C2 change, the dielectric constant around them changes, and as a result, the resonance angle of the surface plasmon resonance phenomenon changes, and the intensity of the reflected light reflected thereby changes. Therefore, if information on the change in the intensity of the reflected light of the portion related to the selected individual living cells C1 and C2 is calculated, the activity of the external stimulus for the individual living cells C1 and C2 is analyzed based on the information. be able to.

In addition, the waveform generation unit 36 uses the waveform data to be measured as an extraction means to determine the characteristics of the temporal change pattern of the dielectric constant observed when the living cells to be examined are exposed to an external stimulus. You may make it extract. In this way, it is possible to analyze living cells using the characteristics of the temporal change pattern of the dielectric constant as an index. The process performed by the waveform generation unit 36 at this time is an extraction process for extracting characteristics of the temporal change pattern of the dielectric constant of the living cells to be examined.

Conventionally, diagnosis and analysis of malignant tumors, etc. are based on visual observation, X-ray, CT (Computed Tomography), or image information by ultrasound, and finally, a tissue structure using a pathological tissue specimen is observed microscopically. It was done based on that. Sometimes, in addition to these, information on the presence or absence of gene abnormality in the suspected tissue or the expression of a marker substance related to cancer may be added.

Diagnosis / analysis of malignant tumors by such macroscopic observation, X-ray, CT, or ultrasound was all obtained by extracting from stationary cells and the structure of the tissue constructed by the cells. This is a method for analyzing abnormalities of specific components. In addition, the presence or absence of expression of a specific gene or other substance can be grasped by a concept slightly different from structural disturbance, but after a biological tissue or cell suspected of having cancer is chemically fixed, In terms of analyzing the amount of quantity, it is still an analysis of the structure of cells or living tissues at a certain moment.

However, the essence of malignant tumors lies in the disordered proliferation and metastasis of cells. In the diagnosis / analysis methods described above, all methods merely observe the possibility of potential functions such as malignant tumors and the resulting structural disturbance. Therefore, the ability to comprehensively and directly analyze the property of cells to proliferate and metastasize as a function of individual living cells means that the diagnosis and analysis of malignant tumors, etc. will be greatly improved. To do.

As a result of intensive studies by the present inventors, normal cells and cancer cells exhibit characteristics of temporal change patterns of different resonance angles with respect to EGF (epidermal growth factor) stimulation by an SPR device. It was elucidated. Furthermore, histopathologically, it was shown that even the same type of cancer or cancer cell line can exhibit different temporal changes in resonance angle characteristics in response to EGF stimulation. In the human hemangiosarcoma cell line, EGFR (epidermal growth factor receptor, Epidermal Growth） Factor 発現 Receptor), which is not expressed in normal vascular component cells, is expressed. Similarly, it has been elucidated that it shows the characteristics of the change pattern with time of different resonance angles.

Therefore, the waveform generation unit 36 analyzes the living cells that are the object of the subject, using the extracted characteristic of the change pattern of the dielectric constant over time as an index. More specifically, the waveform generation unit 36 extracts the characteristics of the measured change pattern of the dielectric constant of living cells over time, for example, monophasic, biphasic, triphasic or other atypical patterns. It is determined which of the conditions is met. The waveform generation unit 36 may perform a more detailed analysis of living cells by comparing with a characteristic of a temporal change pattern of a predetermined dielectric constant. The characteristics of a predetermined dielectric constant change pattern over time include, for example, normal cells (cells without abnormality) that have been measured in advance, and various cancer cells (epithelial / It means the characteristics of the time-dependent change pattern of the dielectric constant of various tumor cells (non-epithelial / fluid), various cancer cells (epithelial malignant tumors) or various cancer cell lines.

For example, the waveform generation unit 36 determines whether or not the living cell that is the subject of the subject is a normal cell, whether or not the target living cell is a cancer cell or a cancer cell, or in the case of a cancer cell Diagnosis / analysis of cancer cells of such types and cell lines (for example, gastric cancer cells, prostate cancer cells, hemangiosarcoma cells (see Examples described later), etc.), etc. The diagnosis / analysis result, that is, the information on the characteristics of the extracted change pattern of the dielectric constant with time (information on the state of the living cells) is sent to the display unit 23. The display unit 23 displays live cell state information sent from the waveform generation unit 36.

The waveform generation unit 36 may further include a database in which conditions are stored. In this case, for example, the waveform generation unit 36 performs the determination by comparing the conditions stored in the database with the extracted characteristics. Such conditions include, for example, the presence or absence of local maximum and local minimum values of the change pattern, the order of local maximum and local minimum values, the initial value, the maximum and minimum values, the range of time for taking local maximum and local minimum values, Conditions for identifying the characteristics of the change pattern, such as the sign and size of the change rate, the change rate of the change rate, etc., can be set.

FIG. 10 is a flowchart showing an example of a cell activity analysis method. As shown in FIG. 10, for example, when analysis of a living cell of a subject is started, an exposure test is performed (step S11). During the exposure test, the dielectric constant is measured (step S12).

Next, the characteristics of the dielectric constant change pattern over time are extracted (step S13). As described above, for example, the extracted characteristics are monophasic, biphasic, triphasic, or other atypical patterns. It is determined which condition is met (step S14). The extraction and the determination thereof are performed in the waveform generation unit 36 as analysis means. Thereafter, the state of the cell with the characteristics of the time-dependent change pattern of the dielectric constant of the extracted characteristics (for example, monophasic, biphasic, triphasic or other atypical patterns) is the object of the subject. The state of the living cell is analyzed and sent to the display unit 23 for display (step S15).

In the present embodiment, it may be used as an analysis system for cancer cells or an analysis system for cancer cells (for example, gastric cancer cells, prostate cancer cells or angiosarcoma cells).

Thus, according to the present embodiment, the state of cells can be comprehensively and directly analyzed at the level of individual living cells. In particular, normal cells and cancers can be obtained only by dynamically analyzing the stimulation response patterns of cells without fixing cells or tissues as in the prior art and evaluating malignant tumors as a potential. The ability to analyze the types of cells, cancer types and cancer cell lines is extremely high in technical value.

In the above embodiment, the display unit 23 and the operation unit 24 correspond to the selection unit, but the present invention is not limited to this. For example, as shown in FIGS. 4 (A) to 4 (C) and FIGS. 5 (A) to 5 (C), the luminance of the region where the living cells are present is the luminance of the region where the living cells are not present. Alternatively, the image processing unit 22 may automatically select a living cell to be measured as a selection unit by using the fact that it is larger than that. The image processing unit 22 extracts a point where a change in spatial luminance is equal to or greater than a predetermined threshold, that is, a change point (edge) in the captured reflection intensity image, and sets the image data surrounded by the edge. May be selected as a measurement target.

In the above embodiment, the optical system of the Kretschmann arrangement in which the reflection surface of the laser beam is the interface F between the metal thin film 5, the glass substrate 4, and the metal thin film 5, is not limited to this. An optical system with an otto arrangement may be employed. In this case, the interface F needs to be disposed at a distance substantially in contact with the nanometer order such that near-field light (evanescent light) is generated with respect to the metal thin film 5.

Further, the metal thin film 5 does not need to be provided on the entire surface of the imaging field, and as shown in FIG. 11, small metal thin films 15 may be arranged on the glass substrate 4 in a matrix, for example. In this case, different live cells may be arranged on each metal thin film 15. In this way, the response of various living cells to the same external stimulus can be measured at a time.

In the above embodiment, the stimulus applying means is the flow cell 9 and the liquid supply unit 10, but the present invention is not limited to this.

A droplet discharge device having a nozzle for dropping a droplet containing an antigen that gives an external stimulus to living cells on the metal thin film 5 may be used as the stimulus applying means. In this case, a plurality of nozzles may be provided, a cluster of living cells (for example, a collection of 1 to 100 living cells) may be arranged, and droplets containing different antibodies or the like may be discharged to each cluster.

Furthermore, in the cell activity analysis method according to the second aspect of the present invention, a cell activity analyzer 100 that omits the flow cell 9 and the liquid supply unit 10 is used. For example, a liquid known in the art such as a pipette or an injector. An embodiment in which a person operates the drop ejection device to give an external stimulus is also possible.

As described above, according to the cell activity analysis apparatus 100 and the cell activity analysis method according to the present embodiment, each living cell is based on the temporal change (change before and after applying the stimulus) of the calculated reflected light intensity. And / or the properties of a part of individual living cells can be evaluated. As a result, the properties of individual living cells can be analyzed and isolated, living cells with certain activities can be screened, specific biomolecules related to cell activity (external stimuli) can be screened, It can be used for various cell studies such as investigating in which part of live cells activation occurs.

The cell activity analysis apparatus 100 and the cell activity analysis method according to the present embodiment can be used for medical diagnosis apparatuses, diagnosis methods, and the like. For example, it can be used for a rapid allergic reaction test of living cells (blood or biopsy material) collected from a living body. It is also possible to stimulate a lesion cell such as cancer with a growth factor or the like and quickly determine whether it is a normal cell or a malignant cell. Furthermore, analysis of the required drug amount that varies depending on the individual (analysis of drug reactivity of peripheral blood cells (lymphocytes, basophils, eosinophils, antigen-presenting cells, etc.) for each individual) can be performed. In addition, it can be used for analysis of drugs that cause drug allergy.

Various reactions occur when living cells are stimulated by changes in any substance or physical condition (eg, aggregation / association of receptor molecules and related intracellular signaling molecules, phosphorylation, dephosphorylation, or membrane) Intracellular translocation including translocation). According to this embodiment, it is possible to analyze what kind of activity change is involved in these reactions.

In particular, in the cell activity analyzer 100 and the cell activity analysis method according to the present embodiment, it is possible to analyze individual living cells and / or a part of each living cell, and thus the above-described sensitivity is higher than that in the past. Such diagnosis, evaluation or analysis can be performed.

In addition, the cell activity analyzer 100 and the cell activity analysis method of the present embodiment can also be used as a high-throughput screening apparatus for clinical diagnosis, for example, a high-throughput allergy diagnosis apparatus. For example, first, a solution containing basophils is obtained from blood collected from a living body using micro magnetic beads or the like (others may be any if including living cells). The basophil solution is described in a multi-well chamber 40 as shown in FIG. 12A (for example, in which wells are vacated in a matrix so that the injected solution is separated) at the time of discharging an external stimulus. It is injected by a droplet discharge device such as that described above or pipetting.

Furthermore, an allergen administration multi-chamber 41 designed to fit the multi-well chamber 40 into which the basophil solution is injected is also prepared. The allergen administration multi-chamber 41 is also configured as a droplet discharge device as described above, and different allergens are preferably administered simultaneously to each well into which blood has been injected. Therefore, the cell activity analyzer 100 and the cell activity analysis method of this embodiment are used. An image captured by the imaging unit 7 of the cell activity analyzer 100 described above is displayed on the display unit 23 of the computer 8. Then, an image as shown in FIG. 12B is displayed, and a highly reliable diagnosis can be performed in a shorter time than a conventional diagnosis method. On the contrary, the allergen may be the same and the specimen from which the basophil solution is collected may be different. Such an apparatus can be used as various high-throughput screening apparatuses such as a high-throughput cancer diagnostic apparatus in addition to an allergy diagnostic apparatus.

Hereafter, the terms used in this specification will be explained.

As used herein, the term “cell” is defined in the same way as the broadest meaning used in the field, and may be any kind or animal cell. Moreover, it may be a naturally occurring cell or an artificially modified cell (for example, a fused cell or a genetically modified cell). “Live cell” refers to a living cell. Of these, cells derived from humans (Homo sapience) are preferred, and mast cells, keratinocytes, human basophils or human B cells are preferred, but not limited thereto.

As used herein, “predetermined magnification” means the magnification of an intensity image by a magnifying optical system such as the objective lens 6. The predetermined magnification needs to be a magnification at which the image data sampled by the image acquisition means can be identified by individual living cells. The predetermined magnification is, for example, 2 to 40 times as described above.

As used herein, “external stimulation” refers to binding of a ligand to a cell surface receptor (for example, a biomolecule such as an antigen described in the present embodiment), environmental changes such as temperature or pH, or mechanical It means a stimulus or an electrical stimulus, and includes all stimuli that act on the activity of a cell (for example, activation of an information transmission system in the cell). Of these, ligand binding to cell surface receptors is preferred, and external stimulation with cytokines is more preferred.

The cytokines in this specification include all types of cytokines known in the art. For example, interleukin, chemokine, interferon, hematopoietic factor, cell growth factor and the like can be mentioned, among which cell growth factor is preferable. Examples of the cell growth factor include EGF, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) or transforming growth factor (TGF) described above. Of these, EGF is most preferred. The term “exposure” means that each external stimulus as described above is given to living cells by an appropriate exposure method.

As used herein, “characteristic of a change pattern with time” means a characteristic of a change rate with time. Specifically, the initial value, the maximum value and / or the minimum value, the time from the exposure to an external stimulus until reaching the maximum value and / or the minimum value, and the change over time are monophasic, biphasic, It means characteristics such as compatibility or other atypical patterns (see Examples). In addition, the “dielectric change over time” or “dielectric change over time pattern” used in this specification depends on changes in the dielectric constant in addition to changes in the dielectric constant with time. The change of the rate of change accompanying the time change of the value (for example, the refractive index or the resonance angle) is also included.

“Analysis” of a living cell in this specification can be defined as including various meanings such as evaluation, identification, classification and diagnosis of a living cell.

In this specification, “extraction” of the characteristics of the temporal change pattern is the characteristics of the measured temporal change pattern among the characteristics of the temporal change pattern as described above. Detection, determination, and / or determination.

In this specification, expressions such as “having”, “including” or “containing” also include the meaning of “consisting of” or “consisting of”.

Hereinafter, examples using the cell activity analyzing apparatus 100 and the cell activity analyzing method according to the present embodiment will be described in detail, but the examples do not limit the present invention.

Example 1
The cell activity analyzer 100 includes a diode laser light source 1 having a wavelength of 630 nm, a polarizing plate 2, a glass substrate 4 on which a metal thin film 5 (50 nm) is deposited, and a prism 3 (S-LAL-10, refractive index = 1.72), A thermostat, an objective lens 6 (4 ×), and a CMOS camera (imaging unit 7) were used. The image obtained by the CMOS camera was subjected to luminance analysis using Image-Pro (manufactured by Media Cybernetics) corresponding to the image processing software program executed by the computer 8 according to the above embodiment.

First, RBL-2H3 cell (Rat Basophilic Leukemia cell, rat basophilic leukemia cell line) is used as a living cell, and DNP-HSA (Dinitrophenyl-Human Serum Albumin (manufactured by Sigma-Aldrich Japan, Tokyo, Japan)) antigen as an external stimulus. An example in which the change in the dielectric constant of individual RBL-2H3 cells was examined will be described. Since RBL-2H3 cells have granules containing histamine in the cells and express IgE receptors on the cell surface, the cells can be activated by antigen-IgE stimulation.

First, RBL-2H3 cells were cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal calf serum (FCS), 100 U / ml penicillin and 100 μg streptomycin, and trypsin was added on the day before the experiment. Used to recover. The collected RBL-2H3 cells were cultured overnight (37 ° C.) on a sensor chip (gold thin film-deposited glass substrate) in the presence of 50 ng / ml anti-DNP-IgE (Sigma-Aldrich Japan, Tokyo, Japan). ).

Next, the chip was mounted on the cell activity analyzer 100 described above, and a running buffer (PIPES buffer) was run. Thereafter, DNP-HSA (50 ng / ml) was injected, and the running buffer was allowed to flow as it was. Using the cell activity analyzer 100, an intensity image of the reflected light magnified by the objective lens (4 times) was obtained every 10 seconds. It was imaged with a CMOS camera. Furthermore, luminance analysis based on the time change with individual living cells as a selected region was performed from the image by Image-Pro. As a control, imaging and luminance analysis were also performed for those in which DNP-HSA was not injected under the same conditions.

4A to 4C are RBL-2H3 cells not stimulated with DNP-HSA, and FIGS. 5A to 5C are CMOS cameras of RBL-2H3 cells stimulated with DNP-HSA. The image at is shown. As shown in FIGS. 4A to 4C, the refractive index of each RBL-2H3 cell did not change for 20 minutes when not stimulated with DNP-HSA (antigen). However, as shown in FIG. 5 (A) to FIG. 5 (C), it clearly increased when stimulated with DNP-HSA.

6A is from the images in FIGS. 4A to 4C, and FIG. 6B is from the images in FIGS. 5A to 5C. Five RBL-2H3 cells are removed. Changes in luminance values (refractive index) measured using Image-Pro and average values thereof are plotted every 10 seconds. The lower line in the graph of FIG. 6B shows the time during which stimulation is performed by DNP-HSA. For the same graph described below, the line indicates the stimulation time. Also in the graph of FIG. 6, as described above, the refractive index of RBL-2H3 cells hardly changed during 20 minutes not stimulated with DNP-HSA, and obviously increased after stimulation with DNP-HSA.

From these results, according to the cell activity analyzer 100 according to the present embodiment, the activity of external stimulation (stimulation by, for example, DNP-HSA (antigen)) on a plurality of living cells based on time change is arbitrarily determined. It was found that each individual living cell can be analyzed without labeling with any of the above substances. Furthermore, as shown in FIG. 6 (B), even the same type of cells and the cells to which the same external stimulus is applied, the intensity of the reflected light, that is, the degree of the reaction of the living cells due to the external stimulus is different. This is because the present invention, which can analyze the activity of external stimuli for each individual living cell, can detect with higher sensitivity than the conventional method of evaluating the average value of the stimulus response to a plurality of living cells. Is shown.

As described in detail in the embodiment, according to the cell activity analyzer 100 according to the present embodiment, it is possible to analyze a dielectric constant of a partial region of the cell, that is, a reaction in (a plurality of different) regions in one cell. It is. 9C is similar to FIGS. 4A to 4C, FIG. 5A to FIG. 5C, FIG. 6A, and FIG. 6B in DNP-HSA. This is an example in which the dielectric constant of RBL-2H3 cells, i.e., the cell response, was analyzed, and the activity of external stimuli and the cellular response were analyzed for a plurality of different regions within one cell.

The results of the examples with other types of live cells or external stimuli are briefly described below.

First, an example using PAM212 cells and A431 cells as living cells and EGF (Epidermal® Growth Factor) (10 ng / ml, manufactured by R & D® system, Minneapolis, MN) as external stimuli will be briefly described. Since PAM212 cells (mouse keratinocyte cell line) express EGF receptor on the cell surface, activation of cells by EGF stimulation is possible. Since A431 cells (Human epithelial carcinoma cell line, human squamous cell carcinoma line) also highly express the EGF receptor on the cell surface, cells can be activated by EGF stimulation. The method for culturing live cells, imaging, and luminance analysis are the same as those in the RBL-2H3 cells described above, and will not be described.

FIG. 13A is an example of an image of a reflection intensity image when a PAM212 cell is stimulated and FIG. FIG. 14A is a graph showing an example of a temporal change in the intensity of reflected light to be measured when the PAM212 cell is stimulated, and FIG. Each of Track 1 to Track 5 shown in FIG. 13 (A) and FIG. 13 (B) shows five living cells or living cell groups selected at random, and the time change of the intensity of these reflected lights is shown in FIG. It is graphed in FIG. 14 (A) and FIG. 14 (B).

Note that Track 6 is a background, that is, a place where there are no living cells, and is used for correcting information related to a change in intensity of reflected light to be measured. The thick lines in FIGS. 14A and 14B indicate average values. As shown in FIG. 13 (A), FIG. 13 (B), FIG. 14 (A), and FIG. 14 (B), activation by stimulation can be observed in any kind of living cells, It has been found that the intensity of the reflected light (ie, the dielectric constant) may decrease after activation by stimulation depending on the type of cell or stimulation, compared to before stimulation. Furthermore, as shown in FIGS. 14 (A) and 14 (B), both cells have a similar graph pattern, but both cells can be distinguished from the intensity of the reflected light. Was also shown. In addition, the measurement target is a large number of living cells as shown in FIGS. 13 (A), 13 (B), 14 (A), and 14 (B) from a part of one living cell as described above. It has also been found that selection and measurement can be made up to the area of the cell group where the cells have gathered.

Furthermore, RBL-2H3 cells bound with anti-DNP-mouse IgE and unbound RBL-2H3 cells as living cells, and PMA (Calbiochem, California, which induces activation of DNP-HSA and RBL-2H3 cells as external stimuli) An example using the state of San Diego is briefly described. In addition, about the culture | cultivation of live cells, imaging, and a brightness | luminance analysis method, it abbreviate | omits similarly.

FIG. 15 is a view showing a sensor chip on which RBL-2H3 cells bound with anti-DNP-mouse IgE and RBL-2H3 cells not bound are arranged. RBL-2H3 cells binding anti-DNP-mouse IgE are circular cells previously cultured in Hydrocell (Cellcellse Inc, Tokyo, Japan) to bind IgE. RBL-2H3 cells not bound to anti-DNP-mouse IgE are spindle shaped cells.

FIG. 16 is a graph showing an example of a temporal change in intensity of reflected light when stimulated with DNP-HSA and PMA in the sensor chip shown in FIG. a is a graph of RBL-2H3 cells bound with anti-DNP-mouse IgE, and b is a graph of RBL-2H3 cells not bound with anti-DNP-mouse IgE. FIGS. 17A to 17C are examples of temporal changes in the image of the reflection intensity image when the sensor chip shown in FIG. 15 is stimulated with DNP-HSA and PMA.

As shown in FIG. 16, after stimulation of DNP-HSA, only RBL-2H3 cells bound with anti-DNP-mouse IgE were activated, and RBL-2H3 cells not bound with anti-DNP-mouse IgE were stimulated with PMA. It turns out that it is activated. Similarly, in FIGS. 17 (A) to 17 (C), only DNP-HSA stimulation at 20 min brightly images mainly circular cells (indicated by arrows in the figure), and at 40 min after PMA stimulation. Spindle-shaped cells are also brightly imaged (indicated by arrows in the figure). Note that graphs a and b in FIG. 16 show average values of changes over time in the intensity of reflected light of both cells indicated by these arrows. From the results shown in FIGS. 16 and 17 (A) to 17 (C), when live cells having different shapes are mixed and the activity of the external stimulus for each live cell is different, each of them is bothered. It was shown that it is possible to analyze the temporal change of the image of each reflection intensity image without isolating each shape of living cells.

Next, an example using RBL-2H3 and RBL-3D4 cells as living cells and DNP-HSA and anti-human IgE antibodies as external stimuli will be briefly described. In addition, about the culture | cultivation of live cells, imaging, and a brightness | luminance analysis method, it abbreviate | omits similarly. RBL-3D4 cells are cells established by the present inventors using genetic engineering techniques for RBL-2H3 cells, and express human IgE receptors. Since RBL-2H3 cells are rat-derived cells, only IgE derived from rats and mice can bind. On the other hand, since RBL-3D4 cells also express human IgE receptor, human-derived IgE can be bound and stimulated with anti-human IgE antibody to activate the cells.

Here, a method for establishing RBL-3D4 cells will be briefly described. First, using human high affinity IgE receptor (FcεRI) α-subunit ｃＤＮＡ and rat γ-subunit ｃＤＮＡ cDNAs, human IgE with human α-subunit as the extracellular region and γ-subunit as the transmembrane region and intracellular region Receptor chimeric protein cDNA was prepared. The prepared cDNA was incorporated into a pEF-BOS expression vector and introduced into RBL-2H3 cells together with a neomycin resistance vector using electroporation. Transduced cells were selected by culturing in the presence of neomycin. Receptor expression was confirmed by flow cytometry using a monoclonal antibody (CRA-1) that recognizes the human IgE receptor. The human IgE receptor-expressing cells established in this way were designated as RBL-3D4 cells.

FIG. 18 is a diagram showing a sensor chip in which RBL-2H3 cells and RBL-3D4 cells are arranged. RBL-2H3 cells are spindle-shaped cells bound with anti-DNP-mouse IgE (anti-DNP mIgE), and RBL-3D4 cells are round-shaped cells bound with human IgE antibody (hIgE). FIG. 19 is a graph showing an example of temporal changes in the intensity of reflected light when the sensor chip shown in FIG. 18 is stimulated with DNP-HSA and an anti-human IgE antibody (manufactured by BETYL, Montgomery, Texas). a is a graph of RBL-2H3 cells, and b is a graph of RBL-3D4 cells.

20 (A) to 20 (C) are diagrams illustrating an example of temporal change in the image of the reflection intensity image when the sensor chip shown in FIG. 18 is stimulated with DNP-HSA and anti-human IgE. The arrow in FIG. 20 (B) shows spindle-shaped RBL-2H3 cells stimulated and activated by DNP-HSA, and the arrow in FIG. 20 (C) is a circle stimulated and activated by anti-human IgE antibody. Shaped RBL-3D4 cells are shown. Graphs a and b in FIG. 19 show the average values of the temporal changes in the intensity of the reflected light of both cells indicated by these arrows. As shown in FIGS. 19 and 20A to 20C, stimulation with DNP-HSA activated only RBL-2H3 cells bound with anti-DNP-mouse IgE, and stimulation with anti-human IgE antibody. It can be seen that only RBL-3D4 cells bound with human IgE antibody are activated. From these results, it was proved that cells having specific IgE antibodies to complementary antigens can be identified by using the cell activity analyzer 100 and the cell activity analysis method according to the present embodiment.

Next, an example using RBL-2H3 cells and A431 cells having anti-DNP-mouse IgE as living cells and DNP-HSA and EGF as external stimuli will be briefly described. In addition, about the culture | cultivation of live cells, imaging, and a brightness | luminance analysis method, it abbreviate | omits similarly.

FIG. 21 is a diagram showing a sensor chip in which RBL-2H3 cells and A431 cells (and A431 cell group (A431 cluster)) are arranged. RBL-2H3 cells are spindle-shaped cells with anti-DNP-mouse IgE, and A431 cells are circular cells. FIGS. 22A and 22B are graphs showing an example of a temporal change in the intensity of reflected light when stimulated with DNP-HSA and EGF in the sensor chip shown in FIG. As shown in FIG. 22, stimulation with DNP-HSA and EGF was performed simultaneously. FIG. 23A and FIG. 23B are diagrams illustrating an example of a temporal change in an image of a reflection intensity image when stimulated with DNP-HSA and EGF in the sensor chip illustrated in FIG.

FIG. 23 (A) shows an image (cont) before giving a stimulus, and FIG. 23 (B) shows an image while giving a stimulus with DNP-HSA and EGF 30 minutes later. Moreover, the arrow in a figure shows five cells selected at random among each cell. A triangular arrowhead indicates RBL-2H3 cells, and a square shape indicates A431 cells. In FIG. 22A, a is a graph showing changes in these five RBL-2H3 cells, and b is a graph showing changes in five A431 cells. As shown in FIG. 22A, it can be seen that even in the same type of cells, there is a difference in the temporal change of the intensity of the reflected light, but a and b are clearly different graph patterns.

In FIG. 22 (B), a represents the average value of changes in RBL-2H3 cells, b represents the average value of changes in A431 cells, c represents the average value of RBL-2H3 cells and A431 cells, and d represents The average value in the whole image is shown. Here, in the graph of a (RBL-2H3 cell average), the intensity of the reflected light remains increased over time. On the contrary, the graph of b (A431 cell average) is lower than that before stimulation after a certain amount of time has passed. Therefore, the change of the graph of d which is the average value of the whole image has become weak.

From these results, when using the cell activity analyzer 100 and the cell activity analysis method according to the present embodiment, co-culture is performed in a state where living cells having different properties are mixed, and a plurality of stimuli are simultaneously applied. Even when given, it was found that a unique signal pattern related to the dielectric constant of individual living cells could be observed. That is, it was shown that the type and nature of the cells can be detected with higher sensitivity and speed than the conventional method for evaluating the average value.

(Example 2)
In Example 2, the change in resonance angle over time when EGF stimulation is performed on CHO (Chinese Hamster Ovary) cells in which wild-type human EGFR is forcibly expressed will be described in detail. EGFR is well known to exhibit important functions in the proliferation of various cells in the body and the development / formation of organs, and is also known to be overexpressed in various cancer cells.

First, an empty vector pCMV-Tag4 (manufactured by Stratagene) and a vector in which wild-type human EGFR gene is incorporated into pCMV-Tag4 are cultivated in Ham's F-12 containing 10% fetal calf serum (FCS, Fetal calf serum). Genes were introduced into CHO cells maintained in solution using electroporation. The wild-type human EGFR gene incorporated into pCMV-Tag4 was amplified by PCR using the forward primer and reverse primer of the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. After gene introduction, drug selection was performed in the presence of 10 mg / ml G418. Thereafter, a CHO cell line introduced with an empty vector and a CHO cell line stably expressing wild-type human EGFR at a high level are dropped on the sensor chip so as to be 1.2 × 10 4 cells / 60 μl, and cultured overnight. did.

After culturing, the cells on the sensor chip were perfused with Hepes buffer, stimulated with 10 ng / ml recombinant human EGF (hEGF, manufactured by R & D) for 10 minutes, and changes in the resonance angle over time after stimulation were measured using SPR- It measured using CELLIA (Mortex company make). Detailed measurement methods using the SPR device are described in Hide M., Tsutsui, T., Sato, H., Nishimura, T., Morimoto, K., Yamamoto, S., Yoshizato, K., 2002, Anal. Biochem. 302 (1), 28-37, Yanase, Y., Suzuki, H., Tsutsui, T., Hiragun, T., Kameyoshi, Y., Hide, M., 2007, Biosens. Bioelectron. 22 (6 ), 1081-1086, and Yanase, Y., Suzuki, H., Tsutsui, T., Uechi, I., Hiragun, T., Mihara, S., Hide, M., 2007, Biosens. Bioelectron. 23 (4), 562-567.

FIG. 24 is a graph showing changes in resonance angle over time by EGF stimulation in CHO cells in which wild-type human EGFR according to Example 2 was forcibly expressed. In FIG. 24, the vertical axis indicates the change in resonance angle (Change of AR (Angle of Resonance) (degree)) by the SPR device, and the horizontal axis indicates the time (Time (sec)) from the EGF stimulation. The same applies to FIGS. 27 to 32 and FIG. 33B described later.

As shown in FIG. 24, when compared with the CHO cell line (mock) into which an empty vector was introduced, the wild-type human EGFR-expressing CHO cell line (EGFR-WT) changes with time in different resonance angles with respect to EGF stimulation. It turns out that it shows a pattern. Specifically, it was confirmed that the wild-type human EGFR-expressing CHO cell line exhibits a typical three-phase resonance angle change pattern over time (a fluctuation pattern that rises from the initial level and rises again after rising). It was.

(Example 3)
In this Example 3, an example relating to EGF stimulation in a CHO cell line expressing human EGFR with a mutated ATP binding domain will be described in detail.

First, a mutant EGFR of the ATP binding domain (EGFR-K721M (Chen, WS, Lazar, CS, Poenie, M., Tsien, RY, Gill, GN, Rosenfeld. MG, 1987, Nature ２８ 328 (6133), 820-823). See)) Genes were generated using QuickChange® Site® Directed® Mutagenesis® Kit (Stratagene) and amplified by PCR. The base sequences of the forward primer and reverse primer used in the PCR are shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing. The prepared ATP-binding domain mutant EGFR gene was introduced into CHO cells by electroporation in the same manner as described in Example 1, and drug selection was performed to obtain a stable expression cell line.

Next, the wild-type human EGFR-expressing CHO cell line of Example 2 and the ATP-binding domain mutant EGFR-expressing CHO cell line described above were seeded in a 6-well plate at a concentration of 0.2 × 10 6 / ml. Cultured overnight in Ham's F-12 culture medium containing 1% fetal bovine serum. Thereafter, the sample was prepared by stimulation with 10 ng / ml hEGF (R & D) for 5 minutes, treatment with a cell lysate containing 1% NP-40, and separation by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). .

After separation, transfer to PVDF (polyvinylidenevinylfluoride) membrane (Immobilon-P, manufactured by Millipore), anti-phosphorylation specific EGFR antibody (Cell signaling), anti-EGFR antibody (Cell 抗 signaling) and anti-FLAG antibody Western blotting was performed using (Stratagene). FIG. 25 is a diagram showing the results of Western blotting using anti-phosphorylation specific EGFR antibody, anti-EGFR antibody and anti-FLAG antibody in CHO cells expressing human EGFR with a mutated ATP binding domain according to Example 2. is there.

As shown in FIG. 25, ATP-binding domain mutant EGFR-expressing CHO cells (K721M) were able to confirm the expression of EGFR protein at an equivalent level or higher when compared to wild-type human EGFR-expressing CHO cells (WT). Tyrosine phosphorylation hardly occurred even after stimulation.

Furthermore, the present inventors determined the expression level of EGFR on the surface of each of ATP-binding domain mutant EGFR-expressing CHO cells and wild-type human EGFR-expressing CHO cells using a PE-labeled anti-human EGFR antibody (BD Biosciences). And FACSCalibar (BD Biosciences). FIG. 26 is a diagram showing measurement results of the expression level of EGFR on the surface of CHO cells in which human EGFR having a mutated ATP binding domain according to Example 3 was expressed.

In FIG. 26, the black-colored ones indicate isotype control antibodies (Control), and the solid-line ones indicate the EGFR cell surface expression level (EGFR-WT) in wild-type human EGFR-expressing CHO cells. The dotted line indicates the EGFR cell surface expression level (EGFR-K721M) in the ATP-binding domain mutant EGFR-expressing CHO cells. As shown in FIG. 26, even in ATP-binding domain mutant EGFR-expressing CHO cells, the cell surface expression level was equivalent to the cell surface expression level of wild-type human EGFR-expressing CHO cells.

In addition, the present inventors seeded the aforementioned ATP-binding domain mutant EGFR-expressing CHO cell line and wild-type human EGFR-expressing CHO cell line on a sensor chip, and the next day, 10 ng / ml hEGF (manufactured by R & D) was used. The sample was stimulated for 10 minutes, and the change in resonance angle over time was measured with SPR-CELLIA (Mortex).

FIG. 27 is a diagram showing changes in resonance angle over time by EGF stimulation in CHO cells in which human EGFR with a mutated ATP binding domain according to Example 2 was expressed. As shown in FIG. 27, the wild-type human EGFR-expressing CHO cell line (EGFR-WT) showed a typical three-phase change in resonance angle similar to the result of Example 1. However, in the ATP-binding domain mutant EGFR-expressing CHO cell line (EGFR-K721M), the resonance angle was hardly changed by stimulation with EGF.

Example 4
In Example 4, examples relating to changes in resonance angle with respect to EGF stimulation of various cancer cell lines will be described in detail.

In the same manner as in Example 2 and Example 3 described above, gastric cancer cell lines (MKN-1, MKN-7 and MK28) and prostate cancer cell lines (DU145 and LNCap) were dropped on the sensor chip, respectively, and the next day Then, stimulation with hEGF (10 ng / ml, manufactured by R & D) was performed for 10 minutes, and the change in resonance angle over time was measured with SPR-CELLIA (manufactured by Moritex).

FIG. 28 is a graph showing changes in the resonance angle over time by EGF stimulation in the gastric cancer cell line MKN-1 according to Example 4. FIG. 29 is a graph showing changes in resonance angle over time by EGF stimulation in gastric cancer cell line MKN-7 according to Example 4. FIG. FIG. 30 is a diagram showing changes in the resonance angle over time by EGF stimulation in the gastric cancer cell line MK28 according to Example 4. FIG. FIG. 31 is a graph showing changes in resonance angle over time by EGF stimulation in prostate cancer cell line DU145 according to Example 4. FIG. FIG. 32 is a diagram showing changes in resonance angle over time by EGF stimulation in the prostate cancer cell line LNCap according to Example 4.

28 to 32, receptor phosphorylation by EGF stimulation was also confirmed by Western blotting as in the method described in Example 2 (each inset). In FIG. 28 to FIG. 32, the upper graphs show the resonance angles over time when EGF stimulation is performed on each cancer cell line (EGF) and when EGF stimulation is not performed on each cancer cell line (Control). The lower graph shows the difference in the change in the resonance angle over time. Furthermore, the number n of experiments conducted independently is also shown in each figure.

As shown in FIG. 28 to FIG. 30, in the gastric cancer cell lines (MKN-1, MKN-7 and MK28), the biphasic change pattern of resonance angle over time ((1) rises and (2) falls by EGF stimulation. A fluctuation pattern) was observed. Further, as shown in FIGS. 31 and 32, in the prostate cancer cell lines (DU145 and LNCap), the change pattern of the uniphasic resonance angle over time by stimulation with EGF (FIG. 31 shows the change pattern of (1) rise only) In FIG. 32, (2) fluctuation pattern of only falling) was observed. In particular, the prostate cancer cell line LNCap did not contain a phase with increasing resonance angle, unlike other cell lines.

From the results of this Example 4 and the typical three-phase resonance angle change pattern of the wild-type human EGFR-expressing CHO cell line described in Example 1 and Example 2, cancers against normal cells By using the characteristics of the change pattern of the resonance angle of the cell over time, that is, the change pattern of the dielectric constant over time, as well as the type of cancer cell line as well as the cell (for example, monophasic, biphasic, triphasic) It was also confirmed that the comprehensive state of living cells can be diagnosed and analyzed by distinguishing by other atypical patterns or the like.

Also, this result suggests that various cell lines other than gastric cancer cell lines or prostate cancer cell lines have characteristics of a time-dependent change pattern of dielectric constant in each cell line. In addition to the diagnosis / analysis of cancer cells relative to normal cells, the distinction between normal cells and cancer cells, and even the distinction between normal cells and cells with some abnormality, is a characteristic of the temporal change pattern of dielectric constant. This suggests that this is possible.

(Example 5)
In Example 5, an example relating to the relationship between the human hemangiosarcoma cell line and the change in resonance angle with respect to EGFR and EGF stimulation will be described in detail. For more detailed relationship between human vascular sarcoma cells, which are human soft tissue sarcoma cells, and EGFR, see J.-L. Yang, MTHannan, PJCrowe, 2006, European Journal of Surgical Oncology (EJSO) 32, 466-468. Please refer to.

First, a human hemangiosarcoma cell line (ISO-HAS) is suspended in DMEM (Dulbecco's Modified Eagle Medium) containing 15% FCS, seeded on a 6-well plate at a concentration of 0.2 × 10 6 / ml, and overnight. Culture was performed. Thereafter, stimulation with hEGF (final concentration of 100 ng / ml, using R & D) for 0 to 15 minutes, treatment with cell lysate, anti-phosphorylation specific EGFR antibody (Cell signaling) and anti-EGFR antibody ( Western blotting was performed in the same manner as described in Example 3 and Example 4 using Cell Signaling).

In addition, the ISO-HAS cells were seeded on a sensor chip, stimulated with hEGF (10 ng / ml, manufactured by R & D) on the next day, and the resonance angle over time was the same as in Examples 3 and 4 described above. Was measured with SPR-CELLIA (Mortex).

FIG. 33 (A) shows the results of western blotting of the human hemangiosarcoma cell line according to Example 5 using anti-phosphorylation-specific EGFR antibody and anti-EGFR antibody. FIG. 33 (B) is a graph showing changes in the resonance angle over time by EGF stimulation in the human hemangiosarcoma cell line according to Example 5. In FIG. 33 (B), as in FIGS. 28 to 32, the upper graph shows the case where EGF stimulation is performed on ISO-HAS cells (EGF) and the case where EGF stimulation is not performed on ISO-HAS cells. The change in resonance angle over time due to (Control) is shown, and the lower graph shows the difference in change in resonance angle over time.

As shown in FIG. 33 (A), the human hemangiosarcoma cell line (ISO-HAS) according to Example 5 expresses EGFR and phosphorylates EGFR upon stimulation with EGF at a concentration of 100 ng / ml. Was confirmed. In addition, as shown in FIG. 33 (B), in the human hemangiosarcoma cell line (ISO-HAS) according to Example 5, an atypical and temporal change pattern of the resonance angle ((1) rising by EGF stimulation) (2) Random (atypical fluctuation patterns) that fell incompletely and fluctuated up and down were observed.

From the results described in Examples 2 to 4 and the results of the human hemangiosarcoma cell line of this Example 5, a sample containing living cells can be used as an object of analysis of the present invention as it is. The possibility that the contained cells can be evaluated and analyzed is also suggested. Specifically, for example, it is suggested that by collecting a body fluid such as blood from a subject in advance and directly analyzing it with an SPR device, it may lead to a technique that can evaluate whether or not any tumor / cancer cell is included. The

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification are incorporated by reference in their entirety.

This application is based on Japanese Patent Application No. 2010-061710 filed on Mar. 17, 2010 and Japanese Patent Application No. 2010-278131 filed on Dec. 14, 2010. The specification, claims, and entire drawings of Japanese Patent Application No. 2010-061710 and Japanese Patent Application No. 2010-278131 are incorporated herein by reference.

According to the present invention, there are provided a cell activity analyzing apparatus and a cell activity analyzing method capable of analyzing the activity of external stimuli for individual living cells. With these cell activity analyzers and cell activity analysis methods, it is possible to evaluate and analyze the properties of individual living cells and / or a part of each living cell, or to isolate specific types of living cells. it can. In addition, live cells with a specific activity are screened, specific biomolecules related to cell activity (external stimuli) are screened, and further, in which part of live cells the activation occurs mainly Can be used for research on various cells. Specifically, it can be used for a medical diagnostic apparatus, for example, a high-throughput allergy diagnostic apparatus.

In addition, the present inventors have clarified that normal cells and cancer cells exhibit characteristics of temporal change patterns of different resonance angles with respect to EGF stimulation using a surface plasmon resonance device (SPR device). Furthermore, it was also shown that different cancer and cancer cell line types exhibit different resonance angle characteristics over time for EGF stimulation. In addition, it was confirmed that the human hemangiosarcoma cell line also has a characteristic of a temporal change pattern of different resonance angles with respect to EGF stimulation.

Therefore, according to the method for analyzing living cells according to the present invention, the state of cells can be comprehensively and directly analyzed at the level of individual living cells. In particular, it is a technology that can analyze cell status, cancer cells, types of cancer and types of cancer cell lines without fixing cells or tissues and evaluating malignant tumors as a potential possibility. High value.

DESCRIPTION OF SYMBOLS 1 Light source 2 Polarizing plate 3 Prism 4

Glass substrate

5, 15 Metal thin film 6 Objective lens 7 Imaging part 8 Computer 9 Flow cell 10 Liquid supply part 11 Microscope 21 Image acquisition part 22 Image processing part 23 Display part 24 Operation part 30 Operation content analysis part 31 Measurement Object Extraction Unit 32 Initial Value Holding Unit 33 Dark

Component Extraction Unit

34, 35 Difference Unit 36 Waveform Generation Unit 40 Multiwell Chamber 41 Multichamber 100 Cell Activity Analyzer

Claims

A cell activity analyzer that analyzes the activity of external stimuli on living cells using the surface plasmon resonance phenomenon,
A metal thin film in contact with the living cell on one side;
A refractive optical element having an interface substantially in contact with the other surface of the metal thin film;
Incident means for causing a P-polarized parallel light beam to enter the refractive optical element and to enter the interface at a predetermined incident angle that causes the surface plasmon resonance phenomenon;
A magnifying optical system for enlarging an intensity image corresponding to a two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface to a predetermined magnification;
Imaging means for capturing the intensity image magnified by the magnification optical system;
Image acquisition means for sampling image data of the intensity image captured by the imaging means;
Selecting means for selecting at least a partial image of the living cells as a measurement target from the image data of the intensity image sampled by the image acquisition means;
The brightness value of the measurement target selected by the selection means is extracted, and the reflected light of the measurement target is based on a change in the brightness value of the measurement target before and after the external stimulus is applied to the living cells. A calculating means for calculating information on a change in intensity of
A cell activity analyzer comprising:
The selection means can specify a plurality of measurement objects,
The calculation means extracts a luminance value of each of the plurality of selected measurement objects from the image data of the intensity image, and calculates information regarding a change in intensity of reflected light of the measurement object for each measurement object. ,
The cell activity analyzer according to claim 1.
The selection means selects a plurality of different locations in the same live cell image as the measurement target,
The cell activity analyzer according to claim 2.
The calculation means is configured to calculate the measurement target based on a difference between a luminance value of the measurement target before applying the external stimulus to the living cells and a luminance value of the measurement target after applying the external stimulus. Calculate information about changes in the intensity of reflected light from
The cell activity analyzer according to claim 1.
The calculation means corrects information related to a change in intensity of reflected light of the measurement target based on a luminance value component of a location where the living cells do not exist in the image data of the intensity image.
The cell activity analyzer according to claim 1.
The predetermined incident angle is equal to a resonance angle when the living cell is not in contact with the metal thin film,
The cell activity analyzer according to claim 1.
An analysis means for analyzing the living cells based on information on a change in intensity of reflected light of the measurement target calculated by the calculation means;
The cell activity analyzer according to claim 1.
The analysis means extracts a characteristic of a time-dependent change pattern of the dielectric constant of the living cell to be measured;
The cell activity analyzer according to claim 7.
The analysis means determines whether the time-dependent change pattern of the dielectric constant of the living cell corresponds to a monophasic, biphasic, triphasic or other atypical pattern.
The cell activity analyzer according to claim 8.
A microscope for observing the living cells in contact with the metal thin film from the one surface side;
The cell activity analyzer according to claim 1.
The selection means includes
Display means for displaying an image based on the image data of the intensity image sampled by the image acquisition means;
An operation means for designating at least a partial image of the living cell as the selected measurement object from image data of the intensity image sampled by the image acquisition means by an operation input;
The cell activity analyzer according to claim 1, further comprising:
The metal thin film can be arranged with a plurality of living cell groups including the living cells apart from each other.
The cell activity analyzer according to claim 1.
Further comprising an external stimulus applying means for applying different external stimuli to each of the plurality of living cell groups,
The cell activity analyzer according to claim 12.
A cell activity analysis method for analyzing the activity of external stimuli on living cells using surface plasmon resonance phenomenon,
An arrangement step of arranging the living cells so as to contact one surface of the metal thin film;
An incident step in which a P-polarized parallel light beam is incident on a refractive optical element having an interface substantially in contact with the other surface of the metal thin film, and is incident on the interface at a predetermined incident angle that causes the surface plasmon resonance phenomenon; ,
An enlargement step of enlarging an intensity image corresponding to a two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface to a predetermined magnification by an enlargement optical system;
An imaging step of capturing the intensity image magnified by the magnification optical system;
An image acquisition step of sampling image data of the intensity image imaged in the imaging step;
From the image data of the intensity image sampled by the image acquisition step, a selection step of selecting at least a partial image of the living cell as a measurement target;
The brightness value of the measurement target selected by the selection step is extracted, and the reflected light of the measurement target is based on a change in the brightness value of the measurement target before and after the external stimulus is applied to the living cells. A calculation step for calculating information on a change in intensity of the
Including
A cell activity analysis method comprising analyzing the activity of the external stimulus with respect to at least a part of the living cells, using as an index information related to the change in intensity of reflected light of the measurement target calculated in the calculation step.
In the selection step, a plurality of measurement objects can be specified,
In the calculation step, a luminance value of each of the plurality of selected measurement objects is extracted from the image data of the intensity image, and information regarding a change in intensity of reflected light of the measurement object is calculated for each measurement object. ,
The method for analyzing cell activity according to claim 14.
The cell activity analysis method according to claim 15, wherein, in the selection step, a plurality of different locations in the same live cell image are selected as the measurement target.
In the calculating step, the measurement target is based on a difference between a luminance value of the measurement target before applying the external stimulus to the living cells and a luminance value of the measurement target after applying the external stimulus. The cell activity analysis method according to claim 14, wherein information relating to a change in the intensity of reflected light is calculated.
In the calculation step, information on a change in intensity of reflected light of the measurement target is corrected based on a luminance value component at a location where the living cells do not exist in the image data of the intensity image. The cell activity analysis method according to claim 14.
The cell activity analysis method according to claim 14, wherein the predetermined incident angle is equal to a resonance angle when the living cell is not in contact with the metal thin film.
The selection step includes
A display step of displaying an image based on the image data of the intensity image sampled in the image acquisition step;
By an operation input, an operation step of designating at least a part of the live cell image as the selected measurement object from the image data of the intensity image sampled in the image acquisition step;
The cell activity analysis method according to claim 14, further comprising:
The cell activity analysis method according to claim 14, wherein in the arranging step, a plurality of living cell groups including the living cells are arranged apart from each other.
The cell activity analysis method according to claim 21, wherein the external stimulus is applied to each of the plurality of living cell groups.
A cell analysis method for analyzing the living cells based on information on a change in intensity of reflected light related to the living cells obtained by analysis using the cell activity analysis method according to claim 14.
Based on the information on the change in the intensity of the reflected light of the measurement object, analyzing the living cells by extracting the characteristics of the change pattern of the dielectric constant of the living cells over time,
The cell analysis method according to claim 23.
Determining whether the change pattern of the dielectric constant of the living cell corresponds to a monophasic, biphasic, triphasic or other atypical pattern,
25. The cell analysis method according to claim 24.
Cancer cells are the subject of analysis,
25. The cell analysis method according to claim 24.
Cancer cells are the subject of analysis,
25. The cell analysis method according to claim 24.
The cancer cell is any of a stomach cancer cell, a prostate cancer cell, or an angiosarcoma cell.
The cell analysis method according to claim 27.
Analyzing the living cells exposed to the external stimulus by cytokines;
25. The cell analysis method according to claim 24.
Analyzing the live cells exposed to the external stimulus by EGF;
25. The cell analysis method according to claim 24.