JP2021099234A - Novel drug discovery screening method - Google Patents

Abstract

To provide a novel drug discovery screening method that can estimate the state of a cell without performing genetic analysis.SOLUTION: The novel drug discovery screening method includes: photographing a cell sample with a candidate drug added to a cell sample; sucking cells; performing image processing on the captured image; obtaining morphological information on the shape of the cells contained in the cell sample based on the image processing result; selecting one cell to be sucked based on the morphological information; sucking the selected one cell; analyzing the sucked one cell; extracting characteristic values of acquired molecular information and morphological information based on the analyzed results; creating a database of cell shape for each cell by storing in the database the morphological information, the molecular information and the feature values in association with one another; and estimating the state of the cells in the sample by referring to the database using the morphological information obtained based on the results of image processing on the image taken of the sample.SELECTED DRAWING: Figure 12

Description

The present invention relates to a novel drug discovery screening method.

In the evaluation test for cell stimulation using living cells, the quantification of labeled known genes and proteins and the prediction of the cell state using the change in shape as an index are performed. In an evaluation test for cell stimulation using living cells, for example, a cell population whose morphology has changed due to stimulation is collected and a gene, which is one of the intracellular substances, is analyzed to confirm the detailed state of the cells.

The following devices have been proposed to confirm the detailed state of cells.
For example, a plate containing a sample (cell) is taken out from the image measurement unit and the incubator unit connected integrally to the image measurement unit, sent to the image measurement unit, measured by the image measurement unit, and then sent to the incubator unit. A drug discovery screening device in which the return operation is repeated has been proposed (see, for example, Patent Document 1).

In addition, a cell concentration and purification device having a function of recognizing the shape and fluorescence emission of each cell on an image basis and a function of separating and purifying the target cell by recognizing based on the information of the shape and fluorescence emission. It has been proposed (see, for example, Patent Document 2).

In addition, an image capture of a cell-derived image obtained by irradiating with a cell irradiation means is performed, a two-dimensional Fourier transform is performed on the captured image, and the target cell is based on the spatial frequency distribution of the image converted by the image conversion means. A cell detection device for detecting the above has been proposed (see, for example, Patent Document 3).

Japanese Unexamined Patent Publication No. 2010-210237 Japanese Unexamined Patent Publication No. 2015-0510008 JP-A-2017-108738

However, in the techniques described in Patent Documents 1 to 3, for example, a floating cell population whose morphology has changed due to stimulation is recovered, and the recovered cell population is subjected to analysis such as gene analysis. Then, in the technique described in Patent Documents 1 to 3, it is necessary for the operator to evaluate the state of the cell such as whether the cell is differentiated or undifferentiated by, for example, looking at an image of the cell.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel drug discovery screening method capable of estimating the state of cells without performing gene analysis or the like.

In order to achieve the above object, in the novel drug discovery screening method according to one aspect of the present invention, a sample in which a candidate drug is added to a cell sample is photographed, and cells contained in the cell sample are aspirated and photographed. Image processing is performed on the image, morphological information regarding the shape of the cells contained in the cell sample is acquired based on the result of the image processing, and one cell to be aspirated is selected and selected based on the morphological information. The one cell was aspirated, the aspirated one cell was analyzed, and the feature amounts of the acquired molecular information and the morphological information were extracted based on the analysis result, and the morphological information was extracted for each cell. And the molecular information and the feature amount are stored in a database to create a database regarding the shape of the cells, and the cell sample is a sample in which a candidate drug is added to the cell sample to be screened. Using the morphological information acquired based on the result of image processing on the photographed image, the state of the cells contained in the sample is referred to with reference to the database. To estimate.

Further, in the novel drug discovery screening method according to one aspect of the present invention, the morphological information includes information indicating the position of a cell or the position of a nucleus, and includes a cell area, a nucleus area, an outer circumference length of a cell, and an outer circumference length of a nucleus. It may be at least one of the major axis of the cell, the major axis of the nucleus, the minor axis of the cell, the minor axis of the nucleus, the apex of the cell, the apex of the nucleus, and the like.

Further, in the novel drug discovery screening method according to one aspect of the present invention, the molecular information is at least one of genetic information, protein information, peptide information, amino acid information, lipid information, sugar information, mass information and the like. It may be.

Further, in the novel drug discovery screening method according to one aspect of the present invention, the molecular information may be genetic information, and the feature amount may be a correlation coefficient between the shape information and the genetic information. ..

According to the present invention, it is possible to provide a novel drug discovery screening method capable of estimating the state of cells without performing gene analysis or the like.

It is a block diagram which shows the structural example of the drug discovery screening system which concerns on 1st Embodiment. It is a figure which shows the structural example of the photographing suction part which concerns on 1st Embodiment. It is a figure which shows the image example of the whole sample which concerns on embodiment. It is a figure which shows the image example of the magnifying photography which concerns on embodiment. It is a figure which shows the processing example which sucks a cell. It is a figure which shows the example of the information stored in the DB which concerns on 1st Embodiment. It is a figure which shows the example of the correlation coefficient calculated from the information stored in the DB which concerns on 1st Embodiment. It is a figure which shows the characteristic example of the cell which contains the gene stored in the DB which concerns on 1st Embodiment. It is a figure which shows the example of the characteristic information of a cell with respect to the morphological information stored in the DB which concerns on 1st Embodiment. It is a flowchart of DB creation processing which concerns on 1st Embodiment. It is a flowchart of the evaluation procedure which evaluates a sample using the DB which concerns on 1st Embodiment. It is a schematic flowchart of the drug discovery screening process which concerns on 2nd Embodiment. It is a flowchart of the drug discovery screening process which concerns on 2nd Embodiment. It is a flowchart of the drug discovery screening process which concerns on 2nd Embodiment. It is a flowchart of the selection process of the cell to be aspirated according to 2nd Embodiment. It is a figure which shows the example of the form information which concerns on 2nd Embodiment. It is a flowchart of processing in the modification of 2nd Embodiment. It is a flowchart of the drug discovery screening process which concerns on 3rd Embodiment. It is a flowchart of the drug discovery screening process which concerns on 3rd Embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings used in the following description, the scale of each member is appropriately changed in order to make each member recognizable.

<First Embodiment>
First, the outline of the present embodiment will be described.
In this embodiment, an example in which the drug discovery screening system creates a database will be described. In the present embodiment, the image obtained by taking a sample is image-analyzed to acquire morphological information, and one cell is aspirated to perform gene analysis to acquire genetic information. Then, in the present embodiment, the morphological information and the genetic information are associated with each other and stored in a database (DB), and the feature amount (for example, correlation coefficient) of the information stored in the DB is extracted. Then, in the present embodiment, the extracted feature amount is stored in association with the information stored in the DB.
Then, in the present embodiment, the state of cells (for example, differentiated or undifferentiated) is estimated by referring to the DB created in this way by using the morphological information extracted from the photographed image of the sample. That is, in the present embodiment, the state of cells can be estimated without analyzing the cells contained in the sample with an analyzer.

[Structure example of drug discovery screening system 1]
FIG. 1 is a block diagram showing a configuration example of the drug discovery screening system 1 according to the present embodiment. As shown in FIG. 1, the drug discovery screening system 1 includes a control device 2, an imaging suction device 3 (imaging unit, suction unit), an analysis device 4-1 and an analysis device 4-2, ..., And a DB 5. In the following description, when one of the analysis device 4-1 and the analysis device 4-2, ... Is not specified, it is referred to as the analysis device 4. The drug discovery screening system 1 is provided with at least one analyzer 4.

The control device 2 includes an operation unit 21, a display unit 22, a suction cell selection unit 23, a DB creation unit 24, a feature amount extraction unit 25, an estimation unit 26, and a control unit 27.
The photographing suction device 3 includes a control unit 31, a photographing suction unit 32, and an image analysis unit 33.
The control device 2 and the photographing suction device 3 are connected by a wired line or a wireless line. Further, the control device 2 and the analysis device 4 are connected by a wired line or a wireless line.

[Operations and functions of each component of the drug discovery screening system 1]
Next, the operation and function of each component of the drug discovery screening system 1 will be described.

First, the operation and function of the control device 2 will be described.
The control device 2 controls the photographing suction device 3 and the analysis device 4.

The control unit 27 outputs a photographing instruction for photographing a sample to the photographing suction device 3, and acquires the photographed image and the image processing result for the photographed image from the photographing suction device 3. The image processing result includes position information indicating the position of the cell or tissue in the cell culture well plate 5 (FIG. 2) and morphological information regarding the shape of the cell or tissue. The morphological information includes information indicating the position of a cell or the position of a nucleus. The morphological information includes, for example, cell area, nuclear area, outer peripheral length of cell, outer peripheral length of nucleus, major axis of cell, major axis of nucleus, minor axis of cell, minor axis of nucleus, apex of cell, apex of nucleus. Includes at least one piece of information such as rate. The control unit 27 causes, for example, the display unit 22 to display the captured image and the image processing result. The control unit 27 acquires information indicating whether the cells used in the analysis are differentiated or undifferentiated based on the operation result of the user operating the operation unit 21 based on the displayed image. You may.

The suction cell selection unit 23 determines whether or not there are cells or tissues to be aspirated based on the acquired image processing result, and when it is determined that there are cells or tissues to be aspirated, the imaging suction device 3 is subjected to. Outputs a suction instruction to suck cells or tissues to be sucked. The target to be aspirated is not limited to cells or tissues, but may include surrounding compounds, culture broth, and the like.
The aspirated cell selection unit 23 outputs an analysis instruction for analyzing the aspirated cells to the analyzer 4. The control device 2 acquires the analysis result from the analysis device 4. The analysis result includes molecular information contained in the analyzed cells or tissues. The molecular information is, for example, at least one of genetic information, protein (including antibody, cytokine, enzyme, etc.) information, peptide information, amino acid information, lipid information, sugar information, mass information, and the like. The suction cell selection unit 23 selects the cells to be sucked based on the operation result of the user operating the operation unit 21 by looking at the captured image displayed on the display unit 22. You may.

The DB creation unit 24 associates the morphological information with the genetic information and stores them in the DB 5.

The feature amount extraction unit 25 obtains the feature amount by calculating the correlation coefficient between each item of morphological information (for example, cell area, cell length) and gene information (gene A, gene B). The feature amount extraction unit 25 stores the obtained correlation coefficient in the DB 5 in association with the information stored in the DB 5.
The feature amount extraction unit 25 acquires information indicating whether the cells used for the analysis are differentiated or undifferentiated, based on the correlation coefficient and the information stored in the DB5.
The feature amount extraction unit 25 stores information indicating whether it is differentiated or undifferentiated in the DB 5 in association with the morphological information stored in the DB 5.

The operation unit 21 is, for example, at least one of a touch panel sensor, a keyboard, a mouse, etc. provided on the display unit 22. The operation unit 21 acquires the operation result operated by the user.

The display unit 22 is, for example, a liquid crystal image display device, an organic EL (Electroluminescence) image display device, or the like.

The estimation unit 26 estimates the state of cells (for example, differentiated or undifferentiated) by referring to the information stored in the DB5 with respect to the morphological information of the image in which the sample to which the reagent is attached is taken. The estimation unit 26 displays the estimated estimation result on the display unit 22.

DB5 is a database. DB5 stores morphological information and genetic information in association with each of the analyzed cells. The DB5 stores the morphological information and the genetic information in association with the correlation coefficient between the morphological information and the genetic information. DB5 stores information indicating whether it is differentiated or undifferentiated in association with morphological information. As will be described later, the DB 5 stores the characteristics of the cell containing the gene in association with each gene name.

Next, the operation and function of the photographing suction device 3 will be described.
The control unit 31 controls the photographing suction unit 32 so as to photograph the set sample in response to the photographing instruction output by the control device 2. The image analysis unit 33 performs image processing on the captured image to acquire morphological information. The control unit 31 outputs the image taken by the photographing suction unit 32 and the image processing result including the morphological information acquired by the image analysis unit 33 to the control device 2. The detailed configuration of the photographing suction unit 32 will be described later with reference to FIG.

The image analysis unit 33 performs image processing on the captured image to acquire morphological information. The image processing performed by the image analysis unit 33 is, for example, processing such as binarization, edge detection, feature amount detection, and clustering.

Next, the operation and the outline of the function of the analysis device 4 will be described.
The analysis device 4 analyzes the cells or tissues sucked by the imaging suction device 3. The analyzer 4 is, for example, a DNA (DeoxyriboNucleic Acid) analyzer, an RNA (RiboNucleic Acid) analyzer, a protein analyzer, and the like. The analysis device 4 outputs the analyzed analysis result to the control device 2. The analysis device 4 includes, for example, a transport unit for transporting the sample, a control unit for controlling the transport unit, and the like.

[Other examples of features]
The calculation of the feature amount is not limited to the calculation of the correlation coefficient described above, and statistical methods such as clustering analysis, principal component analysis, and multivariate analysis may be used, and machine learning (Deep Learning, etc.) may be used. You may use it. Further, the feature amount extraction unit 25 may obtain the feature amount after dividing the data for each cell type and the type of the additive reagent before obtaining the feature amount. Specifically, the table of FIG. 6 to be described later may be used separately for each type of cell tumor or reagent. By dividing the data into layers in this way, it is possible to obtain the effect of clarifying the characteristics of the data. Further, the feature amount extraction unit 25 may perform clustering analysis on the information stored in the DB 5 to group the information, and then obtain the correlation coefficient within each group. Specifically, it is possible to clarify where there is a difference between groups by creating a table of FIG. 6 described later from morphological information and genetic information, performing clustering, and calculating the correlation coefficient within the group. You can get the effect you can.

[Analysis target, analysis method]
Here, an example of an analysis target and an example of an analysis method performed by the analysis device 4 will be described. First, an example of the analysis target will be described.
The analyzer 4 analyzes, for example, a compound possessed or produced by the acquired cells. The analyzer 4 analyzes, for example, the compound produced by the acquired surrounding cells. The analyzer 4 analyzes, for example, the compound added to the culture supernatant of the obtained cells. The analyzer 4 analyzes, for example, a compound administered to an individual of the acquired cells or ingested by the individual.

The analysis target is an organic compound or an inorganic compound contained in the compound. In addition, the analysis targets are proteins (including antibodies, cytokines, enzymes, etc.), peptides, amino acids, lipids, sugars, DNA, RNA (including mRNA, ncRNA, rRNA, tRNA, etc.) contained in organic compounds, and screening. It is a target candidate compound or the like. The ncRNA includes lncRNA, siRNA, miRNA, piRNA and the like. The analysis target is salts, metal ions, etc. contained in the inorganic compound.

The analyzer 4 is to perform at least one of mass spectrometry, NMR (Nuclear Magnetic Resonance), liquid chromatography, Raman spectroscopy, infrared spectroscopy, electrophoresis, and elemental analysis, for example. It may be. The mass spectrometry is, for example, an ionization method. The ionization methods include EI (electron ionization), CI (chemical ionization), FAB (fast atom bombardment), ESI (electrospray ionization), and ESI (electrospray ionization). There is a MALDI (matrix-assisted laser desorption ionization) method and the like. Further, in mass spectrometry, two mass spectrometers (MS) may be connected in series and analyzed by using an apparatus having a collision activation chamber between them.

The analyzer 4 uses techniques such as NGS (next generation sequencer), qPCR (quantitative polymerase chain reaction), microarray, digital PCR, and sequencer in the analysis of DNA and RNA. In RNA analysis, there may be a reverse transcription step. Moreover, as the introduction method of mass spectrometry, a method of directly introducing or connecting liquid chromatography may be used.

[Structure example of shooting suction unit 32]
Next, a configuration example of the photographing suction unit 32 will be described.
FIG. 2 is a diagram showing a configuration example of the photographing suction unit 32 according to the present embodiment. As shown in FIG. 2, the imaging suction unit 32 includes an image cell acquisition unit 321, a suction unit 322, an XYZ stage 323, a suction chip 324, a chip rack 325, a sample rack 326, and a cell culture well plate 5.
The image cell acquisition unit 321 includes an imaging unit 3211, a bright field illumination 3212, and an XY stage 3213. In FIG. 2, the plane on which the XYZ stage 323 moves is defined as the xy plane, and the direction perpendicular to the xy plane is defined as the z-axis direction. The left-right direction of the paper surface is the x-axis direction, and the depth direction is the y-axis direction.

[Operations and functions of each component of the photographing suction unit 32]
On the cell culture well plate 5, a sample in which a candidate drug is added to a sample of a plurality of cells 51 is placed. The sample may contain the cell culture solution 52.

The imaging suction unit 32 photographs the entire image or the image of the singular region in the cell culture well plate 5 placed on the XY stage 3213 in response to the imaging instruction of the control unit 31, and the captured image is image-analyzed. Output to unit 33. The cell culture container may be placed on the XY stage 3213, and is not limited to the cell culture well plate 5, but may be a cell culture dish, a cover glass chamber, a petri dish, or the like. The imaging suction unit 32 drives the XY stage 3213 and the suction unit 322 to suck the cells or tissues to be sucked according to the control of the control unit 31. The imaging suction unit 32 drives the XY stage 3213 to store the suction chip 324 containing the sucked cells or tissues in the sample rack 326 under the control of the control unit 31.

The photographing unit 3211 includes, for example, a microscopic optical unit, a confocal scanning unit, a photographing unit, a fluorescence light source, and an objective lens, as described in Japanese Patent Application Laid-Open No. 2019-41691. Further, the control device 2 includes, for example, a suction time setting unit and a suction pressure setting unit, as described in Japanese Patent Application Laid-Open No. 2019-41691. With these configurations, the photographing suction unit 32 can suck cells or tissues from the sample with less stress, similar to the cell suction support device described in Japanese Patent Application Laid-Open No. 2019-41691.

[Determining cells to be aspirated]
Next, an example of a method for determining a cell or tissue to be aspirated by the drug discovery screening system 1 will be described. In the following description, an example of cells as the target of suction and analysis will be described.

First, for example, the operator sets the cell culture well plate 5 on the XY stage 3213. Then, the imaging suction unit 32 outputs information indicating that the cell culture well plate 5 is set on the XY stage 3213 to the control device 2.
When the cell culture well plate 5 is set in the imaging suction unit 32, the control device 2 outputs an imaging instruction for photographing the entire sample to the imaging suction unit 32.

As shown in FIG. 3, the photographing unit 3211 photographs the entire sample under the control of the control unit 31 that has acquired the imaging control instruction. FIG. 3 is a diagram showing an example of an image of the entire sample according to the embodiment. In FIG. 3, the region of reference numeral g101 is, for example, a region of cells in a state in which the effect of the drug is exhibited (cells in the first state). The regions of the symbols g111 and g112 are, for example, regions of cells in a state in which the effect of the drug is not exhibited (cells in the second state). Further, in FIG. 3, the longitudinal direction is the x direction and the lateral direction is the y direction.

The image analysis unit 33 performs image processing on the captured image to acquire information on, for example, the size of cells. The control device 2 selects, for example, cells having a cell area larger than a predetermined value as a suction target. The control device 2 outputs an instruction to take an enlarged photograph of the region (suction candidate) containing the selected cells to the photographing suction device 3. The control unit 31 of the imaging suction device 3 acquires an instruction to take an enlarged photograph of the region containing the selected cells.

As shown in FIG. 4, the photographing unit 3211 photographs at a magnification higher than the magnification when the entire sample is photographed in response to the instruction of the control unit 31. The imaging unit 3211 sets the magnification when photographing the entire sample to, for example, 10 times, and sets the magnification when photographing the suction candidate to, for example, 100 times. When the resolution of the image sensor of the photographing unit 3211 is high, the photographing unit 3211 does not have to take an enlarged photograph. In this case, the image analysis unit 33 may extract an image of the region of the suction candidate region from the entire image and use it.

FIG. 4 is a diagram showing an example of an image of magnified photography according to the embodiment. In FIG. 4, the region of reference numeral g101 and the region of reference numeral g111 are the same as those in FIG. Further, the symbol g121 indicates an image of the cytoplasm (cell membrane substrate), g122 indicates an image of the nucleus, and the symbol g123 indicates the center of the nucleus. Further, in FIG. 4, the longitudinal direction is the x direction and the lateral direction is the y direction. The photographing unit 3211 may also take a picture of the region of reference numeral g112 in FIG. 3 according to the control of the control unit 31. When there are a plurality of suction candidates, the control unit 31 moves the XY stage 3213 in the xy plane to take a picture of the singular region.

The image analysis unit 33 performs image processing on the captured image of the suction candidate. By this image processing, the image analysis unit 33 obtains morphological information of each of the cell position (center coordinates of the nucleus (x1, y1)), the lengths of the long and short sides in the xy plane, and the area of the cell. Further, as described in Japanese Patent Application Laid-Open No. 2019-41691 described above, the image analysis unit 33 acquires the height of the nucleus by moving the objective lens of the photographing unit 3211 in the z direction and taking a picture. The image analysis unit 33 may perform the same processing on the region of the reference numeral g112 in FIG. The control unit 31 outputs the morphological information acquired by the image analysis unit 33 to the control device 2.

The suction cell selection unit 23 of the control device 2 selects the cells to be sucked based on the morphological information. For example, when there are two suction candidates as shown in FIG. 3, the suction cell selection unit 23 may select to suck cells from any one region, or may select to suck cells from both regions. Good. In this way, the aspirated cell selection unit 23 selects the cells to be aspirated. Further, when there are a plurality of suction candidates, the suction cell selection unit 23 sucks from the plurality of suction candidates based on, for example, the shape of the nucleus, the size (area) of the nucleus, the position of the nucleus, the cell cycle state of the cell, and the like. Cells may be selected. The suction cell selection unit 23 outputs information on cells selected as suction candidates (center coordinates of the nucleus, lengths of long and short sides in the xy plane, height in the z direction) to the imaging suction device 3.

[Suction processing]
Next, the suction process performed by the photographing suction unit 32 will be described.
The photographing suction unit 32 attaches one of the plurality of suction chips 324 stored in the chip rack 325 to the suction unit 322 according to the control of the control unit 31.
Next, the photographing suction unit 32 moves the suction unit 322 and the XY stage 3213 according to the control of the control unit 31 to suck the desired cells or tissues.

FIG. 5 is a diagram showing an example of processing for sucking cells. In the example shown in FIG. 5, the position of the tip tip 324a from the bottom surface in the z-axis direction is z3. In this case, the gap between the tip tip 324a and the cell 51 is small, and the chip tip 324a is not stuck in the cell 51. Further, the state of FIG. 5 is a state in which the cells are adhered to the bottom surface 5a of the cell culture well plate 5. Reference numeral 324b indicates the wall of the suction tip 24.

By accurately controlling the z-axis direction of the tip tip 324a at the start of suction as shown in FIG. 5, the control unit 31 can reduce damage to the target when sucking one cell. The control unit 31 obtains the optimum height by changing the height of the objective lens possessed by the photographing unit 3211 in the z-direction, and controls the tip tip 324a in the z-axis direction at the start of such suction. ..

Next, the imaging suction unit 32 stores the suction chip 324 that sucks the cells or tissues at a predetermined position on the sample rack 326 under the control of the control unit 31.
Next, the control unit 31 outputs information indicating that the cells or tissues have been aspirated to the control device 2.

[Analysis operation of analysis device 4]
Next, the operation of the analysis device 4 will be described.
The analysis device 4 acquires information indicating that cells or tissues have been aspirated from the control device 2. The analyzer 4 performs, for example, gene analysis on the cells or tissues sucked by the photographing suction unit 32 by a well-known method. The analysis device 4 outputs the analyzed gene information to the control device 2.

[Example of information stored in DB5]
Next, an example of information stored in the DB 5 will be described.
FIG. 6 is a diagram showing an example of information stored in the DB 5 according to the present embodiment. As shown in FIG. 6, DB5 provides morphological information (cell area [μm ² ] and cell outer peripheral length [μm]) and gene information (gene A [individual], gene B [individual]) for each cell. Associate and memorize. The morphological information shown in FIG. 6 is an example, and the length of the long side of the cell, the length of the short side of the cell, the area of the nucleus, the outer circumference length of the nucleus, the length of the long side of the nucleus, and the short length of the nucleus. It may be the length of the side, the sharpness in the shape of the cell, the mass of the cell, or the like. Further, the gene information may be information on genes contained in aspirated cells, information on proteins, and the like.

Next, the correlation coefficient will be described as an example of the feature amount extracted from the information stored in the DB 5 of FIG.
FIG. 7 is a diagram showing an example of the correlation coefficient calculated from the information stored in the DB 5 according to the present embodiment. As shown in FIG. 7, the feature amount extraction unit 25 of the control device 2 extracts each of the morphological information and the genetic information stored in the DB 5, and calculates the correlation coefficient for each extracted item. The value used for calculating the correlation coefficient is, for example, an average value of each item.

For example, when the correlation coefficient is 0 to 0.2, the feature amount extraction unit 25 determines that there is almost no correlation, and when the correlation coefficient is 0.2 to 0.4, it determines that there is a weak correlation, and the phase relationship. When the number is 0.4 to 0.7, it is determined that there is a slight correlation, and when the correlation coefficient is 0.7 to 1.0, it is determined that there is a strong correlation. In the example shown in FIG. 7, a strong negative correlation was confirmed between the outer peripheral length and the gene B.

As shown in FIG. 8, the DB 5 stores the characteristics of the cell carrying the gene in association with each gene name. FIG. 8 is a diagram showing a characteristic example of a cell having a gene stored in DB5 according to the present embodiment. For example, when gene B is a gene whose expression is confirmed only in undifferentiated cells, DB5 stores the name of gene B in association with undifferentiated as a feature. In the example shown in FIG. 8, differentiated and undifferentiated examples are shown as features, but the information showing the features is not limited to this.

Information indicating other features is, for example, the bonding area. In adherent cells, when the expression of genes expressed in the epithelium such as E-cadherin and Occludin is reduced, the cell adhesion ability is reduced and the cell adhesion area is considered to be small (the morphology is considered to be rounded). The decrease in gene expression of E-cadherin and Occludin is regarded as an index of EMT (epithelial-mesenchymal transition), and is generally considered to be related to development, wound healing, and canceration.

When the correlation as shown in FIG. 7 is obtained, the feature amount extraction unit 25 estimates that the cells having a large outer circumference are differentiated cells. As a result, the DB creation unit 24 stores information indicating that the cell is a differentiated cell in association with the morphological information having a large outer peripheral length. FIG. 9 is a diagram showing an example of cell characteristic information (feature amount) with respect to the morphological information stored in DB5 according to the present embodiment. As shown in FIG. 9, the DB creation unit 24 stores the characteristic information of the cell indicating that the characteristic is differentiated in association with the characteristic of the shape (for example, the outer peripheral length is large).

The control device 2 may learn the characteristics of the gene shown in FIG. 8 and the characteristics for the shape shown in FIG. 9 by, for example, supervised machine learning and store them in the DB 5.
Further, when the control device 2 dispenses the feature quantity from the information stored in the DB 5, the control device 2 does not calculate the correlation coefficient, but machine-learns the stored information to dispense the feature quantity. It may be.

[DB5 creation processing procedure]
Next, an example of the DB5 creation processing procedure will be described. FIG. 10 is a flowchart of the DB5 creation process according to the present embodiment. The operator places the suction tip 324 according to the purpose of suction on the tip rack 325. For example, when aspirating cell components, a nanospray tip is placed, and when a single cell is aspirated, a normal suction tip is placed.

(Step S1) The operator prepares a cell sample (for example, cancer cells). The operator then places the sample in the cell culture well plate 5. Next, the operator sets the cell culture well plate 5 on the XY stage 3213 of the imaging suction device 3. The imaging suction device 3 detects that the cell culture well plate 5 is set on the XY stage 3213. The imaging suction device 3 may detect that the sample has been set, for example, based on the result of the operator operating the operation unit 21 of the control device 2, and detects the sample by the image captured by the imaging unit 3211. Alternatively, it may be detected by a sensor included in the photographing suction unit 32.

(Step S2) The operator adds a candidate compound reagent) to the prepared cell sample. The operator performs, for example, fluorescent staining as needed. The imaging suction device 3 detects that the reagent has been added. The photographing suction device 3 may detect that the reagent has been added, for example, based on the result of the operator operating the operation unit 21 of the control device 2, and detects it by the sensor included in the photographing suction unit 32. You may.

(Step S3) The imaging suction device 3 photographs the entire sample under the control of the control device 2.

(Step S4) The image analysis unit 33 of the imaging suction device 3 performs image processing on the captured image to acquire morphological information.

(Step S5) The suction cell selection unit 23 of the control device 2 determines whether or not there is a cell or tissue to be sucked based on the morphological information. The suction cell selection unit 23 transmits an instruction to magnify and photograph the region of the suction candidate to the photographing suction unit 32, and sucks based on the morphological information included in the result of the imaging suction unit 32 magnifying and image processing. It may be possible to determine whether or not there is a cell or tissue of interest. When the aspirated cell selection unit 23 determines that there are cells or tissues to be aspirated (step S5; YES), the aspirated cell selection unit 23 proceeds to the process of step S6. When the aspirated cell selection unit 23 determines that there are no cells to be aspirated (step S5; NO), the process returns to the process of step S1.

(Step S6) The aspirated cell selection unit 23 determines the cell or tissue to be aspirated. Subsequently, the suction cell selection unit 23 outputs a suction instruction for sucking the determined cells or tissue to be sucked to the imaging suction device 3. Subsequently, the imaging suction device 3 sucks the cells or tissues to be sucked.

(Step S7) The control device 7 outputs an analysis instruction for analyzing the cells or tissues sucked by the analysis device 4. Subsequently, the analyzer 4 acquires genetic information by performing, for example, gene analysis on the aspirated cells or tissues.

(Step S8) The DB creation unit 24 associates the acquired morphological information with the genetic information and stores it in the DB to update the DB information.

(Step S9) The feature amount extraction unit 25 calculates the correlation coefficient from the DB 5 and dispenses the feature amount. Subsequently, the feature amount extraction unit 25 stores the extracted feature amount in association with the information stored in the DB 5. Subsequently, when the calculated correlation coefficient has a strong correlation, the feature amount extraction unit 25 acquires and acquires cell feature information indicating the state of the cell (for example, differentiated or undifferentiated) by referring to, for example, DB5. Cell characteristic information is associated with morphological information and stored in DB5.

[Example of creating a DB for ES cells]
Here, an example of creating a DB of ES cells (Embryonic Stem Cells; embryonic stem cells) will be described.
First, the operator sets ES cell seeding (one type of cell type) on the cell culture well plate 5 as a sample. Next, the worker adds the differentiation-inducing reagent addition (plurality of reagents).
Next, the imaging suction device 3 photographs ES cells. Next, the image analysis unit 33 acquires morphological information by image analysis.

Next, the suction cell selection unit 23 selects cells to be sucked. Next, the imaging suction device 3 sucks one selected cell.
Next, the analyzer 4 acquires gene information by gene analysis. Next, the DB creation unit 24 of the control device 2 updates the DB 5 by associating the morphological information and the genetic information for each cell.
Next, the feature amount extraction unit 25 of the control device 2 calculates a correlation coefficient for each parameter of the morphological information and the genetic information, and stores the calculated correlation coefficient as the feature amount in association with the information stored in the DB 5. ..

[Example of sample evaluation procedure]
Next, an example of an evaluation procedure for evaluating a sample using the DB of the present embodiment will be described.
FIG. 11 is a flowchart of an evaluation procedure for evaluating a sample using the DB according to the present embodiment.

(Step S101) The imaging suction device 3 detects that the cell culture well plate 5 is set on the XY stage 3213, similarly to step S1 (FIG. 10).

(Step S102) The operator adds a candidate compound reagent) to the prepared cell sample. The imaging suction device 3 detects that the reagent has been added, as in step S2 (FIG. 10).

(Step S103) The imaging suction device 3 photographs the entire sample under the control of the control device 2.

(Step S104) The image analysis unit 33 of the imaging suction device 3 performs image processing on the captured image to acquire morphological information.

(Step S105) The estimation unit 26 estimates the state of cells (for example, differentiated or undifferentiated) by referring to the information stored in DB5 using the morphological information. Subsequently, the estimation unit 26 displays the estimation result on the display unit 22.

As described above, in the present embodiment, the image obtained by taking the sample is image-analyzed to acquire the morphological information, and one cell is aspirated to perform the gene analysis to acquire the gene information. Then, in the present embodiment, the morphological information and the genetic information are associated with each other and stored in the DB5, and the feature amount (for example, the correlation coefficient) of the information stored in the DB5 is extracted. Then, in the present embodiment, the extracted feature amount is stored in association with the information stored in the DB5.

As a result, according to the present embodiment, the state of cells (for example, differentiated or undifferentiated) can be determined by referring to the DB (database) thus created by using the morphological information extracted from the photographed image of the sample. Can be estimated. That is, according to the present embodiment, the state of cells can be estimated based on the captured image without performing analysis such as gene analysis on the cells contained in the sample with the analyzer 4. Further, in the prior art, the evaluation of cells has been made by an experienced person by looking at an image taken. On the other hand, according to the present embodiment, since the cell state is estimated based on the image taken by the cell database creation system for the evaluation of the cell or tissue, the evaluation is performed without the evaluation by an experienced person. Can be done.

Further, in the conventional technique, for example, since a floating cell population whose morphology has changed due to stimulation is collected, it is difficult to evaluate one cell in an adhered state. On the other hand, according to the present embodiment, since the state of the cells or tissues contained in the cell sample is estimated from the image obtained by taking the cell sample, it is possible to evaluate one cell in the adhered state. ..

Here, an example of an evaluation method of a comparative example will be described.
It is known that undifferentiated cells are not maintained when undifferentiated ES cells are cultured, and differentiation occurs in some cells. Experienced workers maintain the undifferentiated state of cells by predicting differentiated cells from the appearance of the cells and removing the cells that appear to have differentiated from the incubator. However, the relationship between morphological changes at the single cell level and the undifferentiated state has not been clarified. Therefore, in a device for removing differentiated cells, a method is conceivable in which an experienced person learns the morphology of the differentiated cells by machine learning to perform differentiation and undifferentiated separation. In this case, it is difficult to judge whether the differentiated and undifferentiated classifications taught by the experienced person are correct.

On the other hand, according to the present embodiment, the relationship between the morphological information and the genetic information is stored in DB5 by calculating the correlation coefficient and using the morphological information obtained from the photographed image of the sample. By referring to the DB, morphological features specifically identified in differentiated cells can be found.

<Additional notes>
(1) An imaging unit that photographs a sample in which a candidate drug is added to a cell sample, and
A database that stores information about cells contained in the cell sample,
A suction part that sucks the cells and
An analyzer that analyzes the aspirated cells and
An image analysis unit that performs image processing on the captured image,
Based on the result of the image processing, morphological information regarding the shape of the cells contained in the cell sample is acquired, one cell to be sucked is selected based on the morphological information, and the selected one cell is selected by the suction unit. The one cell that was aspirated and aspirated was analyzed by the analyzer, and the feature amounts of the molecular information and the morphological information acquired based on the analysis result were extracted, and the morphological information and the morphological information were extracted for each cell. A control device that associates the molecular information with the feature amount and stores it in the database.
Cell database creation system equipped with.
(2) The form information is
Contains information indicating the location of cells or nuclei, including cell area, nuclear area, outer circumference of cells, outer circumference of nuclei, major axis of cells, major axis of nucleus, minor axis of cell, minor axis of nucleus, tip of cell. At least one of rate, nuclear apex, etc.
The cell database creation system according to (1).
(3) The molecular information is
At least one of genetic information, protein information, peptide information, amino acid information, lipid information, sugar information, mass information, etc.
The cell database creation system according to (1) or (2).
(4) The molecular information is
It is genetic information
The feature amount is
It is a correlation coefficient between the morphological information and the genetic information.
The cell database creation system according to (1) or (2).
(5) The control device is
Using the morphological information obtained based on the result of imaging and image processing of the sample, the state of the cells contained in the sample is estimated with reference to the database.
The cell database creation system according to any one of (1) to (4).
(6) The procedure in which the imaging unit photographs a sample in which a candidate drug is added to a cell sample, and
The procedure by which the image analysis unit performs image processing on the captured image,
A procedure in which the control device acquires morphological information regarding the shape of cells contained in the cell sample based on the result of image processing and selects one cell to be aspirated based on the morphological information.
The procedure in which the suction unit sucks the selected one cell, and
The procedure for the analyzer to analyze the aspirated cell and
A procedure in which the control device extracts the feature amount of the molecular information and the morphological information acquired based on the analysis result, and
A procedure in which the control device stores the morphological information, the molecular information, and the feature amount in a database for each cell.
How to create a cell database including.

<Second Embodiment>
In the second embodiment, an example in which the drug discovery screening system 1 performs drug discovery screening will be described.

[Explanation of terms in drug discovery screening]
First, terms in screening for drug discovery development evaluation will be explained.
The drug efficacy determination is a process of determining whether or not the drug added to the cell sampling is effective. The morphological evaluation is a process in which an image of a photographed sample is image-processed and the shape of a cell is evaluated based on the result of the image processing. Structural modification is a process of changing a part of the structure of the added drug. The kinetic confirmation is a process for confirming the effect of a drug on cells, such as theoretically not having an adverse effect on normal cells, based on gene analysis or the like. The physical property evaluation is a process for evaluating whether or not there is a problem with physical properties such as a compound being easily decomposed into a toxic substance. Toxicity evaluation is a process of evaluating whether or not a plurality of doses are administered and accumulated in the body, that is, whether or not there is toxicity.
Further, in the present embodiment, for example, a region in which cells or tissues in a state where the effect of the drug does not appear is referred to as a specific region. Alternatively, the singular region may be a region containing a special cell (for example, a cell having a polynuclear cell).

[Outline of the second embodiment]
Next, the outline of this embodiment will be described.
In general drug discovery screening, treatment was performed in the order of morphological evaluation, drug efficacy determination, structural modification, kinetic confirmation, physical characteristic evaluation, and toxicity evaluation. However, in general drug discovery screening, for example, toxic compounds may remain candidates until the evaluation immediately before the clinical trial. A lot of man-hours and costs are spent on the test up to the toxicity evaluation. Therefore, if the development of a drug using the compound is stopped or the drug discovery screening process is restarted in the toxicity test, the developer will suffer a great loss.
Therefore, in the present embodiment, after the drug efficacy is determined, cells in a specific state are aspirated during the drug discovery screening process, and the aspirated cells are analyzed to estimate their kinetics and toxicity. Thereby, in the present embodiment, the efficiency of the drug discovery screening is improved by determining whether or not to proceed with the drug discovery screening process before the drug discovery screening process is performed to the end.

[Outline of drug discovery screening system 1 and outline of each device]
The configuration of the drug discovery screening system 1 according to the present embodiment is the same as that of the drug discovery screening system 1 (FIGS. 1 and 2) of the first embodiment.
The drug discovery screening system 1 takes an image of a sample to which a candidate drug is added to a cell sample in the process of drug discovery screening, analyzes the photographed image using the information stored in DB5, and analyzes cells or cells. Perform tissue aspiration and analysis of aspirated cells or tissues. The drug discovery screening system 1 determines whether or not to proceed with the drug discovery screening process based on the result of analyzing the aspirated cells or tissues using the information stored in the DB5. The target to be aspirated is not limited to cells or tissues, but may include surrounding compounds, culture broth, and the like.

In the present embodiment, the cell culture well plate 5 carries a sample in which a candidate drug is added to a sample of a plurality of cells 51. The sample may contain the cell culture solution 52. Further, in the following description, it is assumed that a dyeing agent for confirming the effect of the drug is added to the sample.

The imaging suction device 3 photographs the entire image or the image of the singular region in the cell culture well plate 5 placed on the XY stage 3213 in response to the imaging instruction of the control device 2. The photographing suction device 3 analyzes the photographed image, and outputs the image analysis result of the image analysis to the control device 2. The imaging suction device 3 sucks the cells to be sucked in response to the suction instruction of the control device 2. The image analysis result includes the captured image and information indicating the shape, size, position, etc. of each cell contained in the image.

The analysis device 4 performs analysis such as gene analysis on the cells or tissues sucked by the photographing suction device 3 and contained in the suction chip 324 stored in the sample rack 326. The analysis method will be described later. The analysis device 4 outputs the analyzed analysis result to the control device 2.

Similar to the first embodiment, the DB5 stores the morphological information and the genetic information in association with each of the analyzed cells. The DB5 stores the morphological information and the genetic information in association with the correlation coefficient between the morphological information and the genetic information. DB5 stores information indicating whether it is differentiated or undifferentiated in association with morphological information. As will be described later, the DB 5 stores the characteristics of the cell containing the gene in association with each gene name.

The control device 2 controls the imaging suction device 3 so as to photograph the entire sample when the cell culture well plate 5 is set in the imaging suction device 3. In the control device 2, the photographing suction device 3 acquires the image processing result for the entire sample. The control device 2 refers to the information stored in the DB 5 with respect to the image analysis result, and determines whether or not there is a singular region. If there is a singular region, the control device 2 indicates to the display unit 22 that the drug discovery screening should be continued. If there is a singular region, the controller 2 estimates the location of the cell or tissue to be aspirated based on the location of that region. The control device 2 outputs a suction instruction to the imaging suction device 3 so as to suck the cells or tissues to be sucked based on the determined position of the cells or tissues.

[Functions and operation of the photographing suction device 3]
Next, the function and operation of the photographing suction device 3 will be described.
First, for example, the operator sets the cell culture well plate 5 on the XY stage 3213. The imaging suction device 3 outputs information indicating that the cell culture well plate 5 has been set to the control device 2.

Next, the imaging suction unit 32 photographs the entire sample in the xy plane of the cell culture well plate 5 in response to the imaging instruction from the control device 2. The photographing suction unit 32 outputs the photographed image to the image analysis unit 33. The image analysis unit 33 performs image analysis on the captured image to analyze the shape of cells contained in the image and the like. The photographing suction device 3 outputs the image analysis result to the control device 2. The image processing result includes position information of the cell or the position of the cell. The position information is, for example, a value of xy coordinates in the xy plane with the center of the cell culture well plate 5 as the origin.

When the singular region exists, the photographing suction unit 32 moves the XY stage 3213 in the xy plane in response to the photographing instruction from the control device 2 to photograph the singular region. The imaging suction unit 32 photographs the singular region at a higher magnification than when photographing the entire sample. The imaging suction unit 32 sets the magnification when photographing the entire sample to, for example, 10 times, and sets the magnification when photographing a singular region to, for example, 100 times. The image analysis unit 33 performs image analysis on the captured image to analyze the shape of cells (cytoplasmic area, nuclear area, nuclear roundness, etc.) contained in the image. The photographing suction device 3 outputs the image analysis result to the control device 2.

When there are cells or tissues to be sucked, the photographing suction unit 32 causes one of the plurality of suction chips 324 stored in the chip rack 325 to the suction unit 322 in response to a suction instruction from the control device 2. Let it be attached. Next, the photographing suction unit 32 moves the suction unit 322 and the XYZ stage 323 in response to the suction instruction from the control device 2 to suck the desired cells or tissues.

Next, the imaging suction unit 32 stores the suction tip 324 that sucks the cells or tissues at a predetermined position on the sample rack 326. Next, the photographing suction unit 32 outputs information indicating that the suction chip 324 that has sucked the cells or tissues is stored in the sample rack 326 to the analysis device 4.

[Functions and operation of image analysis unit 33]
Next, the function and operation of the image analysis unit 33 will be described.

First, the processing for the image of the entire sample will be described.
The image analysis unit 33 acquires an image of the entire sample output by the photographing suction unit 32. Subsequently, the image analysis unit 33 performs image processing on the acquired image of the entire sample. The image processing includes, for example, edge detection processing, feature amount detection processing, clustering processing, and the like. The image of the entire sample is an image of a sample in which a candidate drug is added to a cell sample and the sample is stained with, for example, a dye in order to confirm the effect of the drug. The image analysis unit 33 outputs the image processing result to the control device 2.

Next, the processing for the image of the singular region will be described.
The image analysis unit 33 acquires an image of a singular region output by the photographing suction unit 32. Subsequently, the image analysis unit 33 performs image processing on the acquired image of the singular region. The image processing result includes position information of the cell or the position of the cell. The image analysis unit 33 outputs the image processing result for the image of the singular region to the control device 2. When there are a plurality of singular regions, the image analysis unit 33 selects at least one singular region from the plurality of singular regions and outputs the position of the cell or tissue in the selected singular region to the control device 2. Good.

[Functions and operation of analyzer 4]
Next, the function and operation of the analysis device 4 will be described.
The analyzer 4 stores information such as genetic information, components, and mass for each cell or tissue.

The analysis device 4 performs, for example, gene analysis on the cells or tissues sucked by the imaging suction device 3. The analyzer 4 estimates the pharmacokinetics and toxicity based on the result of gene analysis and the stored information. As a result of estimating the pharmacokinetics and toxicity, the analyzer 4 controls information prompting the discontinuation of the drug discovery screening process by the added drug (compound) when it is estimated that there is a problem in the pharmacokinetics or toxicity due to the added drug. Output to 2. As a result of estimating the pharmacokinetics and toxicity, the analyzer 4 outputs information prompting the continuation of the drug discovery screening process to the control device 2 when it is estimated that there is no problem in the pharmacokinetics and toxicity due to the added drug. The analysis method for cells or tissues will be described later.

[Functions and operation of control device 2]
Next, the function and operation of the control device 2 will be described.
The operation unit 21 detects the operation result operated by the operator and outputs the detected operation result to the control unit 27. The operation result includes, for example, information indicating that the drug discovery screening process is started, the cell culture well plate 5 is set on the XY stage 3213, and the like.

The control unit 27 outputs a photographing instruction to the photographing suction device 3 so as to photograph the entire sample, and acquires the photographed image and the image processing result for the photographed image. The shooting conditions (for example, designation of optical system such as confocal, epi-fluorescence, phase difference, wavelength setting, shooting interval, total shooting time, etc.) are input by the operator by operating the operation unit 21. It may be stored in advance by the control unit 27.

The control unit 27 instructs the imaging suction device 3 to image the specific region based on the information indicating the position of the cell or the cell in the state where the effect of the drug output by the suction cell selection unit 23 is not appearing. Output. The shooting instruction includes information indicating the position of the singular region to be shot.

The control unit 27 outputs a suction instruction to the photographing suction device 3 so as to suck the desired cells or tissues based on the position information of the cells or tissues to be sucked output by the photographing suction device 3. The control unit 27 receives the analysis result for the aspirated cell or tissue output by the analysis device 4, and displays the received analysis result on the display unit 22.

The aspirated cell selection unit 23 includes cells or tissues in a state in which the effect of the drug is exhibited (cells in the first state) in the sample based on the image processing result for the image of the entire sample and the information stored in the DB5. It is determined whether or not the cells or tissues (cells in the second state) in which the effect of the drug is not exhibited are included. The aspirated cell selection unit 23 outputs to the control unit 27 information indicating the position of the cell or the cell in the state where the effect of the drug does not appear. A method for discriminating between cells or tissues in which the effect of the drug is exhibited (cells in the first state) and cells or tissues in which the effect of the drug is not appearing (cells in the second state) will be described later.

The aspirated cell selection unit 23 determines whether or not there are cells or tissues to be aspirated based on the image processing result for the image of the specific region and the information stored in the DB5. When the aspirated cell selection unit 23 determines that there are cells or tissues to be aspirated, it determines the cells or tissues to be aspirated. The aspirated cell selection unit 23 outputs the determined position information of the aspirated cell or tissue to the control unit 27.

The estimation unit 26 refers to the analysis result output by the analysis device 4 and the information stored in the DB 5 to determine whether or not the kinetics and toxicity can be improved when the structure of the added compound is modified. The estimation unit 26 determines whether or not there is a problem with the dynamics based on the result of the dynamics confirmation performed by the analysis device 4. The estimation unit 26 determines whether or not there is a problem with toxicity based on the result of the physical property determination performed by the analysis device 4.

[Aspiration cell judgment]
Next, an example of a method for determining the cells or tissues to be sucked by the control device 2 and the imaging suction device 3 will be described. In the following description, an example of cells as the target of suction and analysis will be described.

The imaging suction unit 32 photographs the entire sample according to the imaging instruction output by the control device 2, for example, as shown in FIG.

The image analysis unit 33 performs image processing such as clustering on the image of the entire captured material. The image analysis unit 33 outputs the image processing result to the control device 2.

Based on the image processing result, the aspirated cell selection unit 23 refers to the information stored in the DB5 and has a region of the cell in which the effect of the drug is exhibited, or a cell in which the effect of the drug is not exhibited. Determine if there is an area of. The aspirated cell selection unit 23 may determine, for example, a region where cell differentiation has stopped for the drug as a region of the cell in which the effect of the drug is exhibited, based on, for example, the shape and state of the cell. Further, the suction cell selection unit 23 may determine, for example, a region where cell differentiation has not stopped for the drug as a region of the cell in which the effect of the drug has not appeared, based on the shape and state of the cell. Good.

When it is determined that there is no region of the cell in which the effect of the drug is exhibited, the aspirated cell selection unit 23 determines that the effect of the drug has not been obtained. Alternatively, the suction cell selection unit 23 has obtained the effect of the drug when the area of the cell region in which the effect of the drug appears is less than the threshold value with respect to the value occupying the area of the entire sample. It is determined that there is no such thing. Alternatively, the suction cell selection unit 23 can obtain the effect of the drug when the area of the cell region in which the effect of the drug appears is equal to or greater than the threshold value with respect to the value occupying the area of the entire sample. Determine that it is.

The aspirated cell selection unit 23 is in a state in which the effect of the drug is not exhibited when the area of the cell region in which the effect of the drug is exhibited is equal to or greater than the threshold value with respect to the value occupying the area of the entire sample. The central position of the nucleus of the cell contained in the area of the cell is output to the control unit 27. In addition, when the suction cell selection unit 23 has a plurality of cell regions in which the effect of the drug does not appear, the aspiration cell selection unit 23 is the nucleus of the cell included in the region of the cell in which the effect of the drug does not appear in the plurality of regions. The center position is output to the control unit 27. The cells existing in the region of the cells in which the effect of the drug is exhibited are the cells in the first state (cells with major proportions). In addition, cells existing in the region of cells in which the effect of the drug does not appear are cells in the second state (cells with minor proportions).

As shown in FIG. 4, the photographing suction device 3 photographs the singular region at a magnification higher than the magnification when the entire sample is photographed in response to the photographing instruction from the control device 2. The imaging suction device 3 also performs imaging of the region of reference numeral g112 in FIG. 3 in response to an imaging instruction from the control device 2.

The image analysis unit 33 performs image processing on the captured image. The image analysis unit 33 obtains the cell position (center coordinates of the nucleus (x1, y1)) and the lengths of the long side and the short side in the xy plane by this image processing. Further, as described in Japanese Patent Application Laid-Open No. 2019-41691 described above, the image analysis unit 33 moves the objective lens of the imaging unit 3211 of the imaging suction device 3 in the z direction to perform imaging, thereby increasing the height of the nucleus. To get the image. The image analysis unit 33 performs the same processing on the region of the reference numeral g112 in FIG. The image analysis unit 33 includes cell information (center coordinates of the nucleus, lengths of long and short sides in the xy plane, height in the z direction) included in the cell region in which the effect of the drug does not appear. The image analysis result is output to the control device 2.

The aspirated cell selection unit 23 selects cells to be aspirated by referring to the information stored in the DB 5 based on the image analysis result output by the image analysis unit 33. For example, when there are two specific regions as shown in FIG. 3, the aspirated cell selection unit 23 may select to inhale cells from any one region, or may select to inhale from both regions. Good. In this way, the aspirated cell selection unit 23 selects at least one aspirated cell. When there are a plurality of specific regions, the suction cell selection unit 23 sucks from the plurality of specific regions, for example, based on the shape of the nucleus, the size (area) of the nucleus, the position of the nucleus, the cell cycle state of the cell, and the like. Cells may be selected.

[Drug discovery screening procedure]
Next, an example of the drug discovery screening processing procedure will be described with reference to FIGS. 12 to 14. FIG. 12 is a schematic flowchart of the drug discovery screening process according to the present embodiment. 13 and 14 are flowcharts of the drug discovery screening process according to the present embodiment. At the time of screening, the operator places the suction tip 324 according to the suction purpose on the tip rack 325. For example, when aspirating cell components, a nanospray tip is placed, and when a single cell is aspirated, a normal suction tip is placed. If the cell size is unknown, a plurality of sizes of suction chips 324 may be placed on the chip rack 325 in advance. Then, based on the result of image processing, the photographing suction device 3 may select the suction tip 324 according to the control of the control device 2. The cell sample to be evaluated is, for example, a cancer cell.

(Steps S101 to S105) The drug discovery screening system 1 performs the processes of steps S101 to S105 of FIG.
(Step S200) The drug discovery screening system 1 performs a screening process (steps S201 to S217 in FIGS. 12 and 13).

(Step S201) The aspirated cell selection unit 23 determines the drug efficacy based on the state of the cells. When the aspirated cell selection unit 23 is determined to be effective as a result of the drug efficacy determination (step S201; effective), the suction cell selection unit 23 proceeds to the process of step S203. When the suction cell selection unit 23 determines that there is no effect as a result of the drug efficacy determination (step S201; no effect), the suction cell selection unit 23 proceeds to the process of step S202.

(Step S202) The control unit 27 displays on the display unit 22 information indicating that the compound determined to have a problem is excluded. Subsequently, the control unit 27 returns the process to step S101.

(Step S203) The aspirated cell selection unit 23 includes cells or tissues in a state in which the effect of the drug appears in the sample with reference to the information stored in the DB5 based on the image processing result for the image of the entire sample. It is determined whether or not the cell or tissue is contained in a state where the effect of the drug is not exhibited. Subsequently, when the aspirated cell selection unit 23 determines that all the cells show the same morphology, the suction cell selection unit 23 proceeds to the process of step S210 (FIG. 14). When the aspirated cell selection unit 23 determines that cells or tissues in a state where the effect of the drug is not exhibited are contained but exists, that is, when it is determined that cells having a partially different morphology are present, step S204 Proceed to the processing of.

(Step S204) The photographing suction unit 32 photographs an image of a singular region in response to a photographing instruction from the control device 2.

(Step S205) The image analysis unit 33 performs image processing on the captured image and outputs the image analysis result to the control device 2.

(Step S206) The aspirated cell selection unit 23 determines the cell or tissue to be aspirated based on the information stored in the DB 5 based on the image processing result. Subsequently, the control unit 27 generates a suction instruction based on the cells or tissues to be sucked determined by the suction cell selection unit 23, and outputs the generated suction instruction to the imaging suction device 3.

(Step S207) The imaging suction unit 32 sucks desired cells or tissues in response to a suction instruction from the control device 2.

(Step S208) The analyzer 4 performs analysis (for example, gene analysis) on the aspirated cells or tissues. The analysis device 4 outputs the analyzed analysis result to the control device 2.

(Step S209) The estimation unit 26 refers to the analysis result output by the analysis device 4 and the information stored in the DB 5 to determine whether or not the kinetics and toxicity can be improved when the structure of the added compound is modified. To do. When the estimation unit 26 determines that the kinetics and toxicity can be improved by structural modification (step S209; can be improved), the estimation unit 26 proceeds to the process of step S210. When the estimation unit 26 determines that the dynamics and toxicity cannot be improved by structural modification (step S209; cannot be improved), the estimation unit 26 proceeds to the process of step S202 (FIG. 13).

(Step S210) The operator performs structural modification on the compound used in step S101. Subsequently, the operator adds the structurally modified compound to the cell sample prepared in step S101. The operator then places the sample in the cell culture well plate 5. The operator performs, for example, fluorescent staining as needed. Next, the operator removes the cell culture well plate 5 used in steps S101 to S105 and S201 to S209 from the XY stage 3213 of the imaging suction device 3. Next, the operator sets the cell culture well plate 5 on which the sample of the cell sample to which the structurally modified compound is added is placed on the XY stage 3213 of the imaging suction device 3. The imaging suction device 3 detects that the cell culture well plate 5 is set on the XY stage 3213 of the imaging suction device 3.

(Step S211) The analyzer 4 confirms the dynamics of the cells contained in the sample by, for example, performing gene analysis. At the time of confirming the dynamics, the control device 2 controls the photographing suction device 3 to suck cells or tissues from the sample.

(Step S212) The estimation unit 26 determines whether or not there is a problem with the dynamics based on the result of the dynamics confirmation performed by the analysis device 4. When the estimation unit 26 determines that there is no problem in the dynamics (step S212; no problem), the estimation unit 26 proceeds to the process of step S213. When the estimation unit 26 determines that there is a problem in the dynamics (step S212; there is a problem), the estimation unit 26 proceeds to the process of step S202 (FIG. 13).

(Step S213) The analyzer 4 evaluates the physical properties of the added compound.

(Step S214) The estimation unit 26 determines whether or not there is a problem with the physical properties based on the result of the physical property evaluation performed by the analysis device 4. When the estimation unit 26 determines that there is no problem with the physical properties (step S214; no problem), the estimation unit 26 proceeds to the process of step S215. When the estimation unit 26 determines that there is a problem with the physical properties (step S214; there is a problem), the estimation unit 26 proceeds to the process of step S202 (FIG. 13).

(Step S215) The analyzer 4 evaluates the toxicity.

(Step S216) The estimation unit 26 determines whether or not there is a problem with toxicity based on the result of the physical property determination performed by the analysis device 4. When the estimation unit 26 determines that there is no problem in toxicity (step S216; no problem), the estimation unit 26 proceeds to the process of step S217. When the estimation unit 26 determines that there is a problem with toxicity (step S216; there is a problem), the estimation unit 26 proceeds to the process of step S202 (FIG. 13).

(Step S217) The control unit 27 displays information indicating that there is no problem in the screening process on the display unit 22. In response to this, clinical trials on humans will be conducted using drugs (compounds). After the treatment, the drug discovery screening process is completed.

As an example of step S209, if the toxicity can be alleviated by a method such as combined use with a drug that suppresses toxicity by the toxicity evaluation by gene analysis, the process proceeds to structural modification in step S210. For example, if it has an effect of inducing inflammation, a drug having an anti-inflammatory effect is used in combination. And if there is no way to mitigate the toxicity, it may be excluded from the candidate drugs.

[Method of selecting cells to be aspirated]
Next, an example of a method for selecting cells to be aspirated when there are a plurality of singular regions will be described.
FIG. 15 is a flowchart of a cell selection process for aspirated cells according to the present embodiment.

(Step S301) The aspirated cell selection unit 23 lists the morphological information of the cells in each specific region and stores it in DB5.

(Step S302) The aspirated cell selection unit 23 selects at least one from a plurality of items of morphological information.

(Step S303) The aspirated cell selection unit 23 sorts the list of morphological information for the selected item.

(Step S304) The aspirated cell selection unit 23 selects a predetermined number of cells based on the sorted result.

FIG. 16 is a diagram showing an example of form information according to the present embodiment. The morphological information is, for example, the area of the cytoplasm, the area of the nucleus, the roundness of the cytoplasm, the kurtosis of the cytoplasm, and the like. As shown in FIG. 16, the DB 5 stores the morphological information in association with the number. The example shown in FIG. 16 is an example, and the morphological information is not limited to this.

In addition, the suction cell selection unit 23 may select cells or tissues to be sucked as in any of the following I to III.
I. When one parameter of the cell to be aspirated is determined The aspirated cell selection unit 23 selects, for example, the nuclear area from the items (parameters). Then, the aspirated cell selection unit 23 sorts the list of morphological information by the nuclear area, and selects, for example, the top 10 cells.

II. When two parameters of cells to be aspirated are determined The aspirated cell selection unit 23 selects, for example, cytoplasmic roundness and nuclear area from among the items (parameters). Then, the aspirated cell selection unit 23 selects cells having a cytoplasmic roundness of 0.8 or less, sorts the list of morphological information by the nuclear area of the selected cells, and selects, for example, the top 10 cells.

III. When the parameter of the cell to be aspirated is unknown The aspirated cell selection unit 23 selects, for example, the nuclear area from the items (parameters). Then, the suction cell selection unit 23 sorts the list of morphological information by the nuclear area, and selects, for example, cells at the 1st, 10th, 20th, and so on. This gives randomness to cell selection. The suction cell selection unit 23 may randomly select 10 cells.
By randomly aspirating and analyzing cells or tissues in this way, it may be possible to detect unexpected reactions regardless of known parameters.

[Analysis target, analysis method]
Here, an example of an analysis target and an example of an analysis method performed by the analysis device 4 will be described. First, an example of the analysis target will be described.
The analyzer 4 analyzes, for example, a compound possessed or produced by the acquired cells. The analyzer 4 analyzes, for example, the compound produced by the acquired surrounding cells. The analyzer 4 analyzes, for example, the compound added to the culture supernatant of the obtained cells. The analyzer 4 analyzes, for example, a compound administered to an individual of the acquired cells or ingested by the individual.

The analysis target is an organic compound or an inorganic compound contained in the compound. In addition, the analysis targets are proteins (including antibodies, cytokines, enzymes, etc.), peptides, amino acids, lipids, sugars, DNA, RNA (including mRNA, ncRNA, rRNA, tRNA, etc.) contained in organic compounds, and screening. It is a target candidate compound or the like. The ncRNA includes lncRNA, siRNA, miRNA, piRNA and the like. The analysis target is salts, metal ions, etc. contained in the inorganic compound.

Next, an example of the analysis method will be described. The control unit 27 selects a device suitable for analysis from the analysis device 4-1 and the analysis device 4-2, ..., Depending on the type of analysis.
The analyzer 4 performs at least one of mass spectrometry, NMR (Nuclear Magnetic Resonance), liquid chromatography, Raman spectroscopy, infrared spectroscopy, electrophoresis, and elemental analysis, for example. There is an ionization method for mass spectrometry. The ionization methods include EI (electron ionization), CI (chemical ionization), FAB (fast atom bombardment), ESI (electrospray ionization), and ESI (electrospray ionization). There is a MALDI (matrix-assisted laser desorption ionization) method and the like. Further, in mass spectrometry, two mass spectrometers (MS) may be connected in series and analyzed by using an apparatus having a collision activation chamber between them.

In addition, the analyzer 4 uses techniques such as NGS (next generation sequencer), qPCR (quantitative polymerase chain reaction), microarray, digital PCR, and sequencer in the analysis of DNA and RNA. In RNA analysis, there may be a reverse transcription step. Moreover, as the introduction method of mass spectrometry, a method of directly introducing or connecting liquid chromatography may be used.

Here, in the prior art, since a plurality of evaluation and confirmation processes are performed in order, for example, when a problem is found in a toxicity test, a large loss of man-hours and costs is required. In addition, in the conventional technique, since the evaluation is performed by averaging a plurality of cells (for example, evaluation is performed by grinding cells), there is a possibility that some cells having no effect on the drug may have a great influence on the prognosis of the patient. Was there.

On the other hand, in the present embodiment, after determining the drug efficacy during the drug discovery screening process, specific cells (cells in the second state, cells of minor population) are sucked and analyzed.

Thus, according to the present embodiment, whether or not the toxicity can be alleviated by structural modification such as combined use with a drug that suppresses toxicity by aspirating cells during the drug discovery screening process and performing, for example, gene analysis. Can be determined. In the present embodiment, it can be estimated that the aspirated cells cannot improve the drug having such an effect by structural modification before structural modification, kinetic confirmation, physical property evaluation, or toxicity evaluation. Then, in the present embodiment, when it is presumed that the improvement cannot be achieved by structural modification in this way, the drug (compound) is excluded, and the subsequent drug discovery screening process is not performed with the compound. Thereby, according to the present embodiment, the drug discovery screening period can be reduced. Further, according to the present embodiment, individual cells can be aspirated and analyzed in the middle of the drug discovery screening process, so that drug discovery screening can be performed even when a plurality of cell samples are mixed. be able to. In addition, according to the present embodiment, individual cells can be aspirated and analyzed during the drug discovery screening process, so that rare reactions of cells and tissues are not overlooked, and problems of kinetics and toxicity are not overlooked at an early stage. Can be found. Further, according to the present embodiment, since individual cells are aspirated and analyzed in the middle of the conventional drug discovery screening process, it is possible to respond without significantly changing the conventional drug discovery screening process. Further, according to the present embodiment, the cells in the second state (cells with minor proportions) can be subjected to, for example, structural modification processing in parallel with processing such as image processing, suction, and analysis. The drug discovery screening period can be reduced.

In the above-mentioned example, the example in which the gene analysis is performed by the molecular analysis in step S208 of FIG. 14 has been described, but the present invention is not limited to this. The molecular analysis may be an analysis method such as mass spectrometry or Western blotting.

Further, after the cells are acquired in step S207 of FIG. 14, not only the sample may be analyzed, but also the cells may be cultured from one cell, and the test may be performed again after cell proliferation.

Moreover, although the example of the cell was described as the target to which the compound (drug) is added, the target to which the compound is added may be a spheroid (cell aggregate), a tissue, a microorganism, a plant or the like.

In addition to screening for evaluating drug efficacy, the method of the present embodiment removes cells in the second state (cells with minor population) and induces differentiation when stem cells are cultured or induced to differentiate. It can also be used to investigate the cause that does not occur and to confirm the bedroom.

Further, the evaluation target of the screening is not limited to the compound (drug), and may be a stimulus such as light.

Further, the target to be aspirated in step S207 of FIG. 14 is not limited to specific (minor) cells and tissues, but as a positive control, major cells (cells in the first state) may also be aspirated and analyzed. Good.

<Modification example>
A modified example of the embodiment will be described below. In the following modified example, an example of performing a combined analysis of morphological (image) information and genetic information will be described. FIG. 17 is a flowchart of processing in the modified example according to the present embodiment. The following processing is performed, for example, in the process of molecular analysis in step S208 of FIG.

(Step S401) The image analysis unit 33 performs image analysis on the captured image of the sample.

(Step S402) The aspirated cell selection unit 23 selects a first cell (a cell in the first state, a cell having a major proportion) based on the image analysis result. Subsequently, the photographing suction device 3 sucks the first cell in response to the suction instruction from the control device 2.

(Step S403) The aspirated cell selection unit 23 selects a second cell (a second state cell, a cell having a minor proportion) based on the image analysis result. Subsequently, the photographing suction device 3 sucks the second cell in response to the suction instruction from the control device 2.

(Step S404) The analyzer 4 performs gene analysis on each of the first cell and the second cell.

(Step S405) The estimation unit 26 compares the results of the gene analysis.

(Step S406) The estimation unit 26 displays the analysis result on the display unit 22.

<Third Embodiment>
In the above-described embodiment, an example of estimating based on the information stored in the DB 5 has been described, but the present invention is not limited to this. The drug discovery screening system 1 may process using the following information in addition to the estimation based on the information stored in the DB 5.

[Structure example of drug discovery screening system 1]
FIG. 1 is a block diagram showing a configuration example of the drug discovery screening system 1 according to the present embodiment. As shown in FIG. 1, the drug discovery screening system 1 includes a control device 2, an imaging suction device 3 (imaging unit, suction unit), an analysis device 4-1 and an analysis device 4-2, ..., And a DB 5. In the following description, when one of the analysis device 4-1 and the analysis device 4-2, ... Is not specified, it is referred to as the analysis device 4. The drug discovery screening system 1 is provided with at least one analyzer 4.

The control device 2 includes an operation unit 21, a display unit 22, a suction cell selection unit 23 (discrimination unit), a DB creation unit 24, a feature amount extraction unit 25, an estimation unit 26, and a control unit 27.
The photographing suction device 3 includes a control unit 31, a photographing suction unit 32, and an image analysis unit 33.
The control device 2 and the photographing suction device 3 are connected by a wired line or a wireless line. Further, the control device 2 and the analysis device 4 are connected by a wired line or a wireless line.

In the present embodiment, in addition to the database of the first embodiment, DB5 is a state in which the effect of the drug when stained with cells or cell types, for example, shape, size, and dye, appears. Stores image features of cells or tissues, such as color, color of cells in a state where the effect of the drug when stained with a dye does not appear.

[Drug discovery screening procedure]
Next, an example of the drug discovery screening processing procedure will be described with reference to FIGS. 18 and 19. 18 and 19 are flowcharts of the drug discovery screening process according to the present embodiment. At the time of screening, the operator places the suction tip 324 according to the suction purpose on the tip rack 325. For example, when aspirating cell components, a nanospray tip is placed, and when a single cell is aspirated, a normal suction tip is placed. If the cell size is unknown, a plurality of sizes of suction chips 324 may be placed on the chip rack 325 in advance. Then, based on the result of image processing, the photographing suction device 3 may select the suction tip 324 according to the control of the control device 2.

(Step S501) The operator prepares a cell sample (for example, cancer cells). Next, the worker prepares a sample in which the candidate compound is added to the prepared cell sample. The operator then places the sample in the cell culture well plate 5. The operator performs, for example, fluorescent staining as needed. Next, the operator sets the cell culture well plate 5 on the XY stage 3213 of the imaging suction unit 32. The control unit 27 detects that the cell culture well plate 5 is set on the XY stage 3213. The control unit 27 may detect based on the result of the operator operating the operation unit 21 of the control device 2, or may detect the image based on the image captured by the photographing unit 3211, and the photographing suction unit 32 has. It may be detected by a sensor.

(Step S502) The photographing suction unit 32 photographs the entire sample in response to an imaging instruction from the control device 2. Subsequently, the image analysis unit 33 analyzes the shape and the like of the captured image by image processing. Subsequently, the image analysis unit 33 outputs the image processing result to the control device 2. Subsequently, the suction cell selection unit 23 performs morphological evaluation with reference to the information stored in the DB 5 based on the image analysis result output by the image analysis unit 33.

(Step S503) The aspirated cell selection unit 23 determines the drug efficacy based on the result of the morphological evaluation. When the aspirated cell selection unit 23 is determined to be effective as a result of the drug efficacy determination (step S503; effective), the suction cell selection unit 23 proceeds to the process of step S505. When the suction cell selection unit 23 determines that there is no effect as a result of the drug efficacy determination (step S503; no effect), the suction cell selection unit 23 proceeds to the process of step S504.

(Step S504) The control unit 27 causes the display unit 22 to display information indicating that the compound determined to have a problem is excluded. Subsequently, the control unit 27 returns the process to step S501.

(Step S505) The aspirated cell selection unit 23 includes cells or tissues in a state in which the effect of the drug appears in the sample with reference to the information stored in the DB5 based on the image processing result for the image of the entire sample. It is determined whether or not the cell or tissue is contained in a state where the effect of the drug is not exhibited. Subsequently, when the aspirated cell selection unit 23 determines that all the cells show the same morphology, the suction cell selection unit 23 proceeds to the process of step S512 (FIG. 19). When the aspirated cell selection unit 23 determines that cells or tissues in a state where the effect of the drug is not exhibited are contained but exists, that is, when it is determined that cells having a partially different morphology are present, step S506 Proceed to the processing of.

(Step S506) The photographing suction unit 32 photographs an image of a singular region in response to a photographing instruction from the control device 2.

(Step S507) The image analysis unit 33 performs image processing on the captured image and outputs the image analysis result to the control device 2.

(Step S508) The aspirated cell selection unit 23 determines the cell or tissue to be aspirated based on the information stored in the DB 5 based on the image processing result. Subsequently, the control unit 27 generates a suction instruction based on the cells or tissues to be sucked determined by the suction cell selection unit 23, and outputs the generated suction instruction to the imaging suction device 3.

(Step S509) The imaging suction unit 32 sucks desired cells or tissues in response to a suction instruction from the control device 2.

(Step S510) The analyzer 4 performs analysis (for example, gene analysis) on the aspirated cells or tissues. The analysis device 4 outputs the analyzed analysis result to the control device 2.

(Step S511) The estimation unit 26 determines whether or not the kinetics and toxicity can be improved when the structure of the added compound is modified by referring to the analysis result output by the analysis device 4 and the information stored in the DB5. To do. When the estimation unit 26 determines that the dynamics and toxicity can be improved by structural modification (step S511; can be improved), the estimation unit 26 proceeds to the process of step S512 (FIG. 19). When the estimation unit 26 determines that the kinetics and toxicity cannot be improved by structural modification (step S511; cannot be improved), the estimation unit 26 proceeds to the process of step S504.

(Step S512) The operator performs structural modification on the compound used in step S501. Subsequently, the operator adds the structurally modified compound to the cell sample prepared in step S501. The operator then places the sample in the cell culture well plate 5. The operator performs, for example, fluorescent staining as needed. Next, the operator removes the cell culture well plate 5 used in steps S501 to S511 from the XY stage 3213 of the imaging suction device 3. Next, the operator sets the cell culture well plate 5 on which the sample of the cell sample to which the structurally modified compound is added is placed on the XY stage 3213 of the imaging suction device 3. The imaging suction device 3 detects that the cell culture well plate 5 is set on the XY stage 3213 of the imaging suction device 3.

(Step S513) The analyzer 4 confirms the dynamics of the cells contained in the sample by, for example, performing gene analysis. At the time of confirming the dynamics, the control device 2 controls the photographing suction device 3 to suck cells or tissues from the sample.

(Step S514) The estimation unit 26 determines whether or not there is a problem with the dynamics based on the result of the dynamics confirmation performed by the analysis device 4. When the estimation unit 26 determines that there is no problem in dynamics (step S514; no problem), the estimation unit 26 proceeds to the process of step S515. When the estimation unit 26 determines that there is a problem in the dynamics (step S515; there is a problem), the estimation unit 26 proceeds to the process of step S504 (FIG. 18).

(Step S515) The analyzer 4 evaluates the physical properties of the added compound.

(Step S516) The estimation unit 26 determines whether or not there is a problem with the physical properties based on the result of the physical property evaluation performed by the analysis device 4. When the estimation unit 26 determines that there is no problem with the physical properties (step S516; no problem), the estimation unit 26 proceeds to the process of step S517. When the estimation unit 26 determines that there is a problem with the physical properties (step S516; there is a problem), the estimation unit 26 proceeds to the process of step S504 (FIG. 18).

(Step S517) The analyzer 4 evaluates toxicity.

(Step S518) The estimation unit 26 determines whether or not there is a problem with toxicity based on the result of the physical property determination performed by the analysis device 4. When the estimation unit 26 determines that there is no problem in toxicity (step S518; no problem), the estimation unit 26 proceeds to the process of step S519. When the estimation unit 26 determines that there is a problem with toxicity (step S216; there is a problem), the estimation unit 26 proceeds to the process of step S504 (FIG. 18).

(Step S519) The control unit 17 displays information indicating that there is no problem in the screening process on the display unit 22. In response to this, clinical trials on humans will be conducted using drugs (compounds). After the treatment, the drug discovery screening process is completed.

[First Example]
In this example, an example using morphological information (cell cycle) and genetic information (comprehensive analysis) will be described. In this example, a case where a drug that stops the cell cycle of cancer cells and suppresses abnormal growth is used as a specific example will be described.

First, the sample is stained with a fluorescent dye for confirming the cell cycle. After that, the suction cell selection unit 23 determines from the fluorescence information whether the cell is a cell having a G1 cycle or a cell other than the G1 phase.
Next, the suction cell selection unit 23 controls so as to suck one G1 phase cell (cell in the first state) as a control sample. The imaging suction device 3 sucks G1 phase cells from a specific region.
In addition, the suction cell selection unit 23 controls so as to suck one cell other than the G1 phase (cell in the second state) as a target sample. The analyzer 4 simultaneously extracts DNA and RNA from the aspirated cells and quantifies the amount of each gene.
Further, the analyzer 4 confirms the difference in gene expression between the control sample and the target sample and the difference in DNA sequence.
From this, according to this example, it is possible to presume the reason why the drug effect was not obtained in some cells. In addition, according to this example, it is possible to confirm the difference in response to the drug due to the difference in cell cycle.

When preparing a cell sample, the efficiency of the test can be improved by seeding a plurality of types of cells (cells having different resources, etc.) in the same well. In the conventional screening method, when a plurality of cells are seeded in the same well, it becomes difficult to separate the cell types, so that it is necessary to test in a different well for each cell type.
On the other hand, in this example, since one cell can be separated and the origin of the cell can be confirmed by analyzing the DNA of the cell, it is possible to analyze with a mixed sample.

In addition, for example, when screening a drug for treating lung cancer, for example, a drug is added to lung cancer cells for testing. In this case, only one type of cell can be seeded in one well, that is, only a sample with a limited condition of either a man or woman with a medical history of country A, B, C and a dosing history of D can be tested in one well. It becomes. When screening hundreds of thousands of compounds, it is not efficient to evaluate the efficacy of hundreds of cells. However, if the evaluation is performed with a biased sample, the efficacy and toxicity will be overlooked.
On the other hand, according to this example, it is possible to realize the drug efficacy evaluation on the minimum scale of a plurality of samples.

[Second Example]
In the second embodiment, an example using morphological information (cell position information) and genetic information (comprehensive analysis) will be described. In this example, as a specific example, a case of confirming the exclusion mechanism of normal cells for eliminating cancer cells will be described.

First, the suction cell selection unit 23 acquires the position information of each cell based on the result of image processing, and determines whether or not it is in contact with the cancer cell.
Next, the suction cell selection unit 23 outputs a suction instruction to the imaging suction device 3 so as to suck one cell that is not in contact with the cancer cell as a control sample.

The imaging suction device 3 sucks one cell in contact with the cancer cell as a target sample.
The analyzer 4 performs RNA-seq on the aspirated cells to quantify the gene amount.
The analyzer 4 confirms the difference in gene expression between the control sample and the target sample.

As a result, according to this example, genes that are specifically increased or decreased in normal cells in contact with cancer cells can be confirmed, and the mechanism of recognition and elimination of cancer cells by normal cells is estimated. can do. In addition, according to this example, changes in gene expression caused by contact with a specific cell type can be confirmed.

The reason for collecting cells in contact with cancer cells in this way is that not only the cancer cells themselves but also the environment (microenvironment) created by normal cells around the cancer cells affects the progression of the cancer. This is because it is believed to give.

When preparing a cell sample, by seeding a plurality of types of cells (epithelial cells, mesenchymal cells, etc.) in the same well, the test can be performed in an environment closer to the living body. In the conventional screening method, when a plurality of cells are seeded in the same well, it becomes difficult to separate the cell types, so basically a single cell type has been used for the test.
On the other hand, in this example, one cell can be separated, and the origin can be confirmed by discriminating cells by morphology and analyzing DNA, so that analysis with a mixed sample is possible.

[Third Example]
In this example, an example using morphological information (cell movement distance) and genetic information (comprehensive analysis) will be described. In this example, as a specific example, a case of confirming an expression gene of a cancer cell migrating in a collagen gel will be described.

The control unit 27 outputs a photographing instruction to the photographing suction device 3 so as to perform time-lapse photography and record the moving distance of each cell.
The aspirated cell selection unit 23 selects cells having a movement distance of the first threshold value or less and cells having a movement distance of the second threshold value or more based on the image processing result. The second threshold value is larger than the first threshold value.
The suction cell selection unit 23 outputs a suction instruction to the imaging suction device 3 so as to suck one cell whose movement distance is equal to or less than the first threshold value as a control sample.

In addition, the imaging suction device 3 sucks one cell as a target sample whose moving distance is equal to or greater than the second threshold value.
The analyzer 4 performs RNA-seq on the aspirated cells to quantify the gene amount.

As a result, according to this example, genes that are specifically increased or decreased in highly mobile cancer cells (highly malignant cells) can be confirmed, which is effective for more highly malignant cells. The drug can be estimated. In addition, according to this example, the relationship between cell migration and gene expression can be confirmed.
The reason for aspirating cells having a large mobility is that cells with a high mobility are considered to be highly malignant cells that easily cause infiltration or metastasis in the field of cancer.

[Fourth Example]
In the fourth embodiment, a case example in which morphological information (morphology of a cell population) and genetic information (comprehensive analysis) are used will be described. In this example, as a specific example, a case where undifferentiated cells are confirmed during culturing of undifferentiated cells will be described.

First, the image analysis unit 33 digitizes the cell density and the smoothness of the contour shape of the colonies formed by the undifferentiated cells by image analysis and stores them in the DB 5 as morphological information.
The aspirated cell selection unit 23 selects cells located in the center of a colony having a high cell density and a smooth contour shape based on the image analysis result. Further, the suction cell selection unit 23 selects cells located in the contour portion of the colony whose cell density is low and the contour shape is not smooth. Further, the aspirated cell selection unit 23 selects cells that are separated from the colony and survive alone.

The suction cell selection unit 23 outputs a suction instruction to the imaging suction device 3 so as to suck one cell as a control sample C, which is located in the center of a colony having a high cell density and a smooth contour shape. Further, the suction cell selection unit 23 outputs a suction instruction to the imaging suction device 3 so as to suck one cell as the target sample A, which is located in the contour portion of the colony having a low cell density and a non-smooth contour shape. The suction cell selection unit 23 outputs a suction instruction to the imaging suction device 3 so as to suck one cell that is detached from the colony and survives independently as the target sample B.

The analyzer 4 performs RT-qPCR (real-time qPCR) on the aspirated cells to quantify the amount of undifferentiated marker gene. RNA-seq (sequence) may be used.
The analyzer 4 confirms the difference in gene expression of samples A, B, and C.

Thereby, according to this example, it can be confirmed whether the entire well including A, B, and C maintains undifferentiated state. In addition, according to this example, changes in the morphology of the cell population due to differences in gene expression can be confirmed.

The outline of the confirmation of undifferentiated state can be estimated from the image information. And gene analysis is required for accurate evaluation. Therefore, in this example, cells are classified according to arbitrary parameters based on the image analysis results, and one cell of each of the different classifications is aspirated to perform comprehensive gene analysis.

In this example, if the correlation between the morphological information and the genetic information is confirmed, the undifferentiated state can be confirmed only from the morphological information.

Although the example of performing gene analysis in the molecular analysis of step S208 of FIG. 14 has been described, multivariate analysis may be performed on the gene analysis result and morphological information. Thereby, according to the present embodiment, there is a possibility that a new correlation between morphology and gene expression can be discovered.

<Additional notes>
(1) The imaging unit performs imaging so as to include a plurality of the cell samples in the sample to which the candidate drug is added to the cell sample.
The discriminating unit performs image processing on the captured image, and based on the result of the image processing, whether or not the cells in the first state are present in the plurality of cell samples, and the cells in the second state are present. Determine if you want to
When the cells in the second state are present, the imaging unit photographs the region in which the cells in the second state are present.
The suction unit sucks the cells in the second state and
The analysis unit analyzes the components of the aspirated cells in the second state,
Based on the analysis result, the control unit determines whether or not to proceed with the drug discovery screening process.
Drug discovery screening method.
(2) The cell in the first state is a cell or tissue in which the effect of the candidate drug appears in the sample.
The drug discovery screening method according to (1), wherein the cells in the second state are cells or tissues in which the effect of the candidate drug does not appear in the sample.
(3) The discriminating unit analyzes the morphology of a plurality of the cells based on the captured image, and determines whether or not the effect of the added candidate drug appears based on the result of the morphology analysis. When the effect of the added candidate drug appears, it is determined whether or not the cells in the first state are present in the plurality of cells, and whether or not the cells in the second state are present (1). ) Or (2).
(4) The components analyzed by the analysis unit include the compound possessed by the aspirated cell, the compound produced by the aspirated cell, the compound produced by the aspirated surrounding cell, and the aspirated compound. Any one of (1) to (3), which is at least one of a compound added to the culture supernatant of a cell, a compound administered to an individual of the aspirated cell, and a compound ingested by the aspirated cell. The drug discovery screening method described in 1.
(5) The drug discovery screening according to any one of (1) to (4), wherein the imaging unit photographs the stained sample so as to include a plurality of the cell samples. Method.
(6) The discriminator analyzes at least one morphology of the area of the cytoplasm of the cell, the area of the nucleus of the cell, the roundness of the cytoplasm of the cell, and the sharpness of the cytoplasm of the cell. The drug discovery screening method according to 3).
(7) The suction unit also sucks the cells in the first state, and the analysis unit analyzes the components of the sucked cells in the second state, and the analysis results for the cells in the first state and the first state. The drug discovery screening method according to any one of (1) to (6), which compares the analysis results for cells in two states.
(8) A drug discovery screening system including an imaging unit, a suction device, an image processing device, a control device, and an analysis device.
The control device controls the imaging of the imaging unit, controls the suction operation of the suction device based on the result of image processing by the image processing device, and drug discovery based on the result analyzed by the analysis device. Determine whether to proceed with the screening process and
In accordance with the control of the control device, the imaging unit photographs a sample in which a candidate drug is added to the cell sample so as to include a plurality of the cell samples, and a second state different from the cells in the first state. When the cells of the above-mentioned second state are present, the region where the cells of the second state are present is photographed.
The image processing apparatus performs image processing on the captured image, and based on the result of the image processing, whether or not the cells in the first state are present in the plurality of cell samples, the second state. Determine if the cells are present
When the cells in the second state are present, the suction device sucks the cells in the second state according to the control of the control device.
The analyzer analyzes the components of the aspirated cells in the second state.
Drug discovery screening system.

A program for realizing all or part of the functions of the control device 2 or the photographing suction device 3 in the present invention is recorded on a computer-readable recording medium, and the program recorded on the recording medium is stored in the computer system. By reading and executing, all or part of the processing performed by the control device 2 or the photographing suction device 3 may be performed. The term "computer system" as used herein includes hardware such as an OS and peripheral devices. Further, the "computer system" shall also include a WWW system provided with a homepage providing environment (or display environment). Further, the "computer-readable recording medium" refers to a portable medium such as a flexible disk, a magneto-optical disk, a ROM, or a CD-ROM, or a storage device such as a hard disk built in a computer system. Furthermore, a "computer-readable recording medium" is a volatile memory (RAM) inside a computer system that serves as a server or client when a program is transmitted via a network such as the Internet or a communication line such as a telephone line. In addition, it shall include those that hold the program for a certain period of time.

Further, the program may be transmitted from a computer system in which this program is stored in a storage device or the like to another computer system via a transmission medium or by a transmission wave in the transmission medium. Here, the "transmission medium" for transmitting a program refers to a medium having a function of transmitting information, such as a network (communication network) such as the Internet or a communication line (communication line) such as a telephone line. Further, the above program may be for realizing a part of the above-mentioned functions. Further, it may be a so-called difference file (difference program) that can realize the above-mentioned function in combination with a program already recorded in the computer system.

Although the embodiments for carrying out the present invention have been described above using the embodiments, the present invention is not limited to these embodiments, and various modifications and substitutions are made without departing from the gist of the present invention. Can be added.

1 ... Drug discovery screening system,
2 ... Control device,
3 ... Shooting suction device,
4-1, 4-2, 4 ... Analytical device,
5 ... Cell culture well plate,
21 ... Operation unit,
22 ... Display,
23 ... Suction cell selection unit,
24 ... DB Creation Department,
25 ... Feature extraction unit,
26 ... Estimator,
27 ... Control unit,
31 ... Control unit,
32 ... Shooting suction part,
33 ... Image analysis unit,
321 ... Image cell acquisition unit,
322 ... Suction part,
323 ... XYZ stage,
324 ... Suction tip,
325 ... Chip rack,
326 ... Specimen rack,
3211 ... Shooting department,
3212 ... Bright field lighting,
3213 ... XY stage

Claims (4)

Take a picture of the sample in which the candidate drug is added to the cell sample,
The cells contained in the cell sample are aspirated and
Image processing is performed on the captured image,
Based on the result of the image processing, morphological information regarding the shape of the cells contained in the cell sample is acquired, one cell to be aspirated is selected based on the morphological information, and the selected one cell is aspirated.
The aspirated one cell was analyzed and
The feature amounts of the molecular information and the morphological information acquired based on the analyzed results are extracted, and the morphological information, the molecular information, and the feature amount are stored in a database for each cell in association with each other. Create a database on the shape of the cells
In the screening sample, which is a sample in which the candidate drug is added to the cell sample to be screened, an image is taken so as to include a plurality of the cell samples.
Using the morphological information acquired based on the result of image processing on the captured image, the state of the cells contained in the sample is estimated with reference to the database.
New drug discovery screening method. The morphological information is
Contains information indicating the location of cells or nuclei, including cell area, nuclear area, outer circumference of cells, outer circumference of nuclei, major axis of cells, major axis of nucleus, minor axis of cell, minor axis of nucleus, tip of cell. At least one of rate, nuclear apex, etc.
The novel drug discovery screening method according to claim 1. The molecular information is
At least one of genetic information, protein information, peptide information, amino acid information, lipid information, sugar information, mass information, etc.
The novel drug discovery screening method according to claim 1 or 2. The molecular information is
It is genetic information
The feature amount is
It is a correlation coefficient between the morphological information and the genetic information.
The novel drug discovery screening method according to claim 1 or 2.

Images