JP7365094B2 - Drug efficacy evaluation method, computer program and recording medium - Google Patents

Description

The present invention relates to a drug efficacy evaluation method for determining the efficacy of a test substance inoculated or administered to cultured cells. The term "medicinal efficacy" as used in this specification is a concept that includes both the positive effects that a test substance has on cells, such as promoting proliferation and improving lesions, and the negative effects such as toxicity that damages cells. Furthermore, hereinafter, "inoculation or administration" will be abbreviated as "inoculation".

In the technical field of drug discovery, which involves developing new drugs, and the technical field of drug efficacy evaluation, which investigates the effects of various substances such as pathogens and the toxins they produce on living organisms, cultured cells are inoculated with test substances, and the subsequent The state of the cells is observed. In visual observation using a microscope or the like, variations in judgment occur due to subjectivity, and the judgment results also vary depending on how the observation field is selected. Therefore, there is a need to establish a quantitative evaluation method that does not rely on subjectivity for the effects of test substances on cells.

Conventional techniques for meeting this purpose include those described in Patent Documents 1 and 2, for example. The technique described in Patent Document 1 uses a biochemical method of measuring the amount of antibodies produced by cells due to the action of pathogenic bacteria. Furthermore, in the technique described in Patent Document 2, quantitative measurement is performed by detecting the amount of fluorescence emitted by cells into which a substance that reacts with an excitation light source is incorporated.

JP2014-202535A Japanese Patent Application Publication No. 2014-117170

Since the above-mentioned conventional techniques use special reagents and light sources for quantitative measurement, advance preparation is required and measurement costs tend to be high. Furthermore, since the cells themselves are modified or destroyed, there is a problem that they are not suitable for observation over time. Therefore, there is a need to establish a method that can objectively evaluate drug efficacy without requiring special preparations for measurement or modification of cells.

This invention has been made in view of the above-mentioned problems, and is a drug efficacy evaluation method for determining the efficacy of a test substance inoculated into cultured cells, which does not require special preparation for measurement or modification of cells. The purpose is to provide technology that allows objective evaluation of drug efficacy.

In order to achieve the above object, one aspect of the present invention includes a step of acquiring a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations; For each of the original images, a step of extracting an object corresponding to the cell in the original image, and calculating the circularity of each of the objects included in the evaluation target area of the original image for each of the original images. This is a drug efficacy evaluation method comprising the steps of determining the average value, and determining the efficacy of the test substance on the cells based on the mode of change in the average value with respect to changes in the concentration of the test substance.

In the invention configured in this manner, the efficacy of the test substance can be quantitatively evaluated by utilizing the fact that the shape of cells present in a sample as single cells or clusters changes depending on the medicinal efficacy of the test substance. That is, when certain types of cells are combined with a test substance, cells or cell aggregates that are generally spherical under normal culture conditions have a property of becoming amorphous due to the drug effect of the test substance. Alternatively, there may be a combination in which cells or cell aggregates, which normally have a shape specific to the cell type, have the property of aggregating into a spherical shape due to the drug effect of the test substance.

For example, in cells whose shape changes from spherical to amorphous due to the medicinal effect of a test substance, the disorder of the cell shape increases as the concentration of the test substance increases. Therefore, in an image of a cell, the circularity of the object corresponding to the cell decreases as the medicinal efficacy increases. On the other hand, in a system whose shape approaches a spherical shape due to its medicinal efficacy, the degree of circularity increases as the medicinal efficacy increases.

From this, it is possible to quantitatively evaluate the magnitude of drug efficacy by imaging samples prepared with various concentrations of the test substance and determining the circularity of objects in the images. As one specific embodiment, in the present invention, for each of a plurality of original images taken with various concentrations of the test substance, the circularity of the object extracted in the evaluation target region in the original image is determined, The drug efficacy is determined based on the relationship between the change in the average value and the change in the concentration of the test substance. Since this quantitatively shows the change in circularity (average value) with respect to the change in concentration, it is possible to objectively judge the drug efficacy based on the change in circularity (average value).

The original image only needs to show the shape of cells or cell aggregates, and may be obtained by bright field imaging without using staining or fluorescent reagents. Therefore, there is no need for equipment for addition of reagents, staining, or fluorescence imaging, and it is possible to reduce the effort and cost required for imaging.

In the drug efficacy evaluation method according to the present invention, at least the step of extracting an object from the acquired original image, the step of calculating the circularity of the object, and the step of determining the efficacy of the test substance on cells are performed using a computer. It is possible to have the person as the execution entity. In this sense, the present invention can be realized as a computer program for causing a computer to execute the above processing. Moreover, it is also possible to realize it as a recording medium that records the computer program.

As described above, according to the present invention, the relationship between the concentration of a test substance and the corresponding change in the shape of cells can be quantitatively shown from an image of a sample obtained by bright field imaging. Based on the results, it is possible to objectively evaluate the efficacy of the test substance on cells.

1 is a flowchart showing a method for evaluating drug efficacy according to the present embodiment. 3 is a flowchart showing a sample preparation process. 3 is a flowchart showing visual field selection processing. FIG. 3 is a diagram schematically showing an example of an image in a processing process. FIG. 3 is a diagram schematically showing an example of a block image that is appropriate as an evaluation target area and a block image that is inappropriate. FIG. 2 is a diagram showing an example of drug efficacy evaluation according to the present embodiment. 2 is a flowchart showing a method for evaluating drug efficacy for unknown samples. FIG. 2 is a diagram illustrating the configuration of an entity that executes drug efficacy evaluation processing according to the present embodiment.

Hereinafter, one embodiment of the drug efficacy evaluation method according to the present invention will be described. Here, as an example of drug efficacy evaluation, a case will be described in which the drug efficacy is evaluated when pertussis toxin produced by Bordetella pertussis is made to act as a test substance on Chinese hamster ovary cells (CHO cells). Note that "medicinal efficacy" as used herein refers to the ability of a test substance to cause some kind of change in cells, and the concept of such change includes both what is favorable and what is unfavorable for cells. Therefore, in this case, the specific morphological changes that pertussis toxin causes on cells correspond to "medicinal effects."

Under normal culture conditions, cells or cell clusters of a certain cell type have a generally circular shape in two dimensions and a roughly spherical shape in three dimensions. However, due to the medicinal effects of pertussis toxin, its shape becomes disordered. In addition, although cells or cell aggregates usually have a shape specific to the cell type, there are cases where the shape becomes more rounded depending on the medicinal effect. Conventionally, the presence or absence of disorder in the shape of cell aggregates has been comprehensively determined by visual observation using a microscope or the like, but variations in the determination due to the subjectivity of the observer and the setting of the observation field are unavoidable. This embodiment solves this problem and enables objective determination.

However, the present invention is not limited to this, and can be applied to various combinations of cells and test substances. In particular, as in the above example, the shape of cells or cell aggregates that are normally approximately circular or spherical is disrupted by the drug effect, or conversely, cells or cell aggregates that normally have a cell type-specific shape are disrupted by the drug effect. In the case of rounding, the drug efficacy evaluation method of the present invention is effective. In the pertussis toxin used in this embodiment, normally squamous cells change into spherical shapes to form cell aggregates.

FIG. 1 is a flowchart showing the drug efficacy evaluation method of this embodiment. Further, FIG. 2 is a flowchart showing the sample preparation process. As shown in FIG. 1, in this drug efficacy evaluation method, a plurality of samples are first bright-field imaged to obtain original images (step S101). Each sample is produced, for example, as follows. As shown in FIG. 2, appropriate medium is injected into a plurality of wells provided in, for example, a well plate (step S201). Cells to be experimented are seeded in each well and cultured under predetermined culture conditions (step S202).

When cells are cultured for a certain period of time under appropriate culture conditions, they proliferate, and some types of cells aggregate with each other to form cell colonies. In the following, unless otherwise specified, the term "cell" refers to both a single cell and a cell colony formed by aggregation of a plurality of cells. Furthermore, the term "cell colony" generally refers to an aggregate of multiple cells, but does not exclude cells existing singly from the concept.

A sample is prepared by inoculating a test substance at a predetermined concentration into each well in which cells are cultured in this manner (step S203). Cells cultured under the same conditions are inoculated with different concentrations of test substances. By varying the concentration of the test substance in each well, multiple samples can be prepared on one well plate. For example, concentrations can be assigned along a dilution series in which the concentration of the test substance changes at a constant rate. Specific numerical examples will be described later.

Bright-field imaging is performed for each of the samples thus prepared, thereby obtaining a plurality of original images corresponding to each sample (step S101). The original image only needs to be able to detect the shape of each cell colony, so prior staining, fluorescence imaging, etc. are not required. The imaging may be performed by imaging a part of the sample in the well using a microscope or the like, but it is more preferable that the entire well be included in the imaging field of view. This is because, due to variations in sample preparation, specifically variations in the distribution of cells at the time of seeding, the subsequent progress of culture, and the way the test substance spreads during injection, drug efficacy may vary depending on location within a single well. This is because the appearance may be different.

When imaging only a portion of the well within the field of view, the image may not represent the typical state of the sample. As described later, in this embodiment, the entire well is imaged at the time of imaging, and an appropriate area is selected from the resulting original image for evaluation, which prevents inappropriate field of view selection. It is possible to prevent misjudgments caused by this.

The processes from the next steps S102 to S105 are executed for each original image captured for each sample. In step S102, objects corresponding to cell colonies are extracted from each original image acquired for each sample. The extraction method is not particularly limited, and various methods of dividing the image into areas corresponding to cell colonies and other areas may be applied, such as known binarization processing such as adaptive thresholding and edge detection. It is possible. Cells and waste products (debris) released from cell colonies during the culture process can also be detected as objects, but these are eliminated as much as possible based on the shape and concentration of the objects, and only objects corresponding to cell colonies are extracted. It is more preferable. Since there are various known examples of such methods, their explanation will be omitted here.

Next, an appropriate field of view is selected based on the distribution state of objects in the image (step S104). As described above, the original image of one well may contain a mixture of regions that are appropriate for evaluation and regions that are inappropriate for evaluation due to variations during sample preparation. Therefore, the original image is divided into several block images, and an appropriate block image is selected from among them as the target for evaluation. Such processing is equivalent to selecting an image captured in a field of view suitable for evaluation from a plurality of microscope images in which different partial regions within the well are respectively captured in the field of view. Therefore, this process is called visual field selection.

In evaluation based on the shape of cell colonies, it is ideal that a plurality of cell colonies of appropriate size are distributed at an appropriate density in the field of view (block image) subjected to evaluation. In order to select a block image that meets these conditions, in this embodiment, the total area of objects occupying the block image and the distance between objects are used as indicators. The ratio of the area occupied by an object within a block image is a concept known as "confluency" in the technical field of cell culture, and is used as an indicator of the progress of culture.

Specifically, even if there are many cell colonies, if each colony is small, the total area will be small; conversely, even if there are few cell colonies, if each colony is large, the total area will be large. Become. In order to contain an appropriate number of cell colonies of appropriate size, the confluency value is preferably within a predetermined appropriate range.

Further, the distance between objects is an index representing whether a plurality of cell colonies are separated from each other or in contact with each other. It is preferable that a plurality of cell colonies be isolated from each other within a block image. This is because when cell colonies are in contact with each other, if they are detected as a single object, the shape of each colony cannot be correctly evaluated, leading to erroneous determination. Furthermore, if the objects are too far apart, the number of objects existing in one block image will decrease, and the accuracy in subsequent statistical processing will decrease. Therefore, there is also an appropriate range for the distance between objects.

FIG. 3 is a flowchart showing the visual field selection process. Further, FIG. 4 is a diagram schematically showing an example of an image in the processing process. The process shown in FIG. 3 is a process for selecting an appropriate field of view based on the above principle. First, the image after object extraction is divided into a plurality of block images (step S301). FIG. 4A shows an example in which an image Ia including a plurality of objects is divided into four parts by broken lines. However, the number of divisions of the original image is not limited to this and is arbitrary.

Then, the position of the center of gravity of the object included in each block image is specified (step S302). The method of determining the center of gravity of an image object is known, and the known method can be applied here as well. Next, Delaunay triangulation is performed on a point set whose elements are the centroids of each object (step S303). Delaunay triangulation is a concept that corresponds to the duality of Voronoi triangulation in which the center of gravity of each object is the generating point, and when applied to the block image of this embodiment, each side of the triangle is the center of gravity of two adjacent objects. Generated to connect between. Therefore, the length of each side represents the distance between the centroids of two adjacent objects.

Image Ib in FIG. 4(b) is the upper right one of the four block images obtained by dividing image Ia in FIG. The sides of triangles generated by triangulation are shown by solid lines. As is clear from this figure, the sides of each triangle connect the centroids of adjacent objects, and the length represents the distance between the centroids. In other words, objects whose centers of gravity are connected as a result of Delaunay triangulation can be uniquely considered to be "mutually adjacent objects." Note that FIG. 4 and FIG. 5, which will be described later, are schematic diagrams for explaining the concept of division, and do not necessarily show correct division results.

For example, objects OB1 and OB2 connected by one side S1 of the Delaunay triangle, and objects OB2 and OB3 connected by side S2, can be regarded as a pair of adjacent objects, and the relationship between these objects is The distances are represented by the lengths of sides S1 and S2, respectively. On the other hand, objects OB1 and OB3 that are not connected by a single edge are not considered to be adjacent to each other.

This is equivalent to considering a pair of objects corresponding to the centroids of Voronoi cells that are in contact with each other as "adjacent to each other" as a result of performing Voronoi division using the centroid of each object as a generating point.

In a state where a plurality of objects are separated from each other and dispersed, the distance between adjacent pairs of objects is relatively large. Further, it is considered that the distances do not differ greatly between each pair of objects if the objects are generally uniformly distributed. In other words, there are appropriate ranges for the length of the sides of the Delaunay triangle and the value of the standard deviation when considering this as a population. In other words, these values are indicators of the degree of object dispersion. Therefore, in addition to the length of each side, the value of the standard deviation with the length of the sides as a population is calculated (step S304).

Further, as described above, in an image of a sample that has been cultured in good condition and is suitable for observation, there is an appropriate range for the ratio of the area occupied by the cell colony region in the image (confluency). Therefore, the ratio of the total area of the object to the area of the block image is also calculated (step S305).

The range that can be considered appropriate is set in advance for the parameters thus obtained, namely the average value of the distance between cell colonies (i.e. the length of the sides of the Delaunay triangle), its standard deviation, and the area ratio of the cell colonies in the block image. be done. A block image that satisfies the conditions specified by them is selected as a block image suitable for evaluation (step S306). The area of the block image selected in this way will be referred to as the "evaluation target area" in the original image.

For example, according to the inventor's knowledge regarding images of cell colonies inoculated with pertussis toxin, the preferred range of confluency values for block images with a side size of approximately 1000 pixels is 14% to 30%, A preferable range of the distance between colonies is 100 pixels to 130 pixels, and a preferable range of the standard deviation thereof is about 50 pixels to 65 pixels. When the values of the parameters determined for one block image are all within these appropriate ranges, the block image can be used as an "evaluation target area."

Naturally, it is conceivable that there are a plurality of block images that satisfy such conditions. In that case, all of them may be set as the evaluation target area, or some of them may be set as the evaluation target area.

FIG. 5 is a diagram schematically showing examples of block images that are appropriate as evaluation target regions and block images that are inappropriate. FIG. 5(a) is an example of a block image suitable as an evaluation target area, and the variation in the side lengths of triangles obtained as a result of Delaunay triangulation is small. On the other hand, in the example of FIG. 5(b), there are sides with widely different lengths, and the variation is large. Such a block image is not suitable as an evaluation target area.

FIG. 5C is a diagram illustrating the result of visual field selection for a certain original image. Here, one original image was divided into 16, and each parameter, that is, the average value of the distance between cell colonies, its standard deviation, and the area ratio of the cell colonies, was determined for each block image after the division. Block images (block Nos. 1 to 8) whose values are all within the above-mentioned appropriate ranges are considered suitable for the evaluation target area. On the other hand, blocks (blocks No. 9 to 16) in which any parameter is outside the appropriate range are considered to be unsuitable for the evaluation target area. Note that the block number (Block No.) is a code added for convenience to distinguish each block image in FIG. do not have.

In this way, even if a block image is extracted from one original image, its suitability as an evaluation target region varies greatly depending on its position. Therefore, when only a part of the visual field randomly or subjectively selected from the entire sample is to be evaluated, an inappropriate selection result may lead to an erroneous determination. In this embodiment, as described above, block images whose values for each parameter have a score of a certain value or higher are candidates for the evaluation target area, so that erroneous determinations due to inappropriate visual field selection can be avoided.

Returning to FIG. 1, the explanation of the drug efficacy evaluation method will be continued. The processes after step S104 are executed on the block image that is the evaluation target area. The objects existing in the evaluation target area are narrowed down (step S104). Objects extracted as corresponding to cell colonies may also include objects that are not suitable for shape evaluation. For example, the larger the number of cells that make up a cell colony, the more the overall outline shape becomes independent of the shape of each individual cell, but in a cell colony that is made up of an extremely small number of cells, the outline becomes smaller depending on the shape of each individual cell. It strongly reflects the shape of. Further, if a plurality of cell colonies are in contact with each other and are detected as a single object as a whole, the outline of the whole will be distorted even if the individual cell colonies are circular, for example.

As described below, in this embodiment, the average value of the circularity of cell colonies is used as an index of drug efficacy. Therefore, as mentioned above, objects corresponding to cell colonies whose shapes greatly deviate from their original shapes due to causes unrelated to drug efficacy become a source of error and must be excluded from evaluation targets. If the type of cells to be experimented with is known, it is possible to estimate in advance the size of individual cells and the appropriate size of a cell colony. Therefore, considering these findings, objects that are clearly too small or too large should be excluded from the target.

By setting an appropriate range for the area of objects and excluding objects whose size deviates from this range, it is possible to narrow down the objects included in the calculation. Here, based on the inventor's knowledge, objects with an area equivalent to 3 cells to an area equivalent to 180 cells are subject to calculation. Extremely small objects or extremely large objects that fall outside this range are excluded from calculation targets. Note that these numerical values can be changed as appropriate.

The circularity of each object narrowed down in this way is determined (step S105). There are various calculation formulas that express how close to a circle an object is, but the basic idea is that when an object is circular, the value is 1, and the further it deviates from the circle, the smaller the value becomes. It is something. In this embodiment, the following formula:
C=4πS/L ² ... (Formula 1)
The circularity C expressed by is adopted. Here, S is the area of the object and L is its perimeter. This is one of the most basic definitions of various types of circularity.

In addition to this, circularity is expressed by the area of the object and the longest radius, circularity is expressed by the area of the object and the length of the major axis when the object is approximated as an ellipse, and the area of the object and the area of the convex hull. It is also possible to express the degree of convexity expressed by the ratio of . However, according to the knowledge obtained by the inventor through various experiments, the method according to the above (Formula 1) is superior in that calculation is simple and good results can be obtained.

From the circularity values determined for each object, an average value of circularity within the evaluation target area is further determined (step S106). The shapes of objects are individually different, and by statistically processing them, it is possible to suppress judgment errors caused by individual variations in shape. In order to enhance its effectiveness, it is desirable that a sufficient number of objects be included within the evaluation target area. The visual field selection process described above also serves this purpose. Hereinafter, the average value of circularity obtained in this manner will be referred to as "average circularity."

In a cell colony that becomes flat or irregularly shaped under a good culture environment, the average circularity increases to a relatively large value (close to 1) when the test substance has a medicinal effect. On the other hand, if the test substance has no medicinal effect and there is no change in shape, the average circularity will be a low value. Since the greater the medicinal efficacy, the greater the average circularity, the average circularity can be an indicator of the magnitude of the medicinal efficacy. For the same test substance, the higher the concentration of the sample, the greater the medicinal efficacy and the more pronounced the increase in average circularity. On the other hand, if the average circularity does not change much depending on the concentration, it is considered that the medicinal efficacy of the test substance is small. Conversely, in cell colonies that aggregate roundly due to drug efficacy, the degree of circularity increases with the magnitude of the drug efficacy of the test substance. From these facts, it is possible to objectively determine whether or not the test substance has a medicinal effect based on the change in the average circularity with respect to the concentration of the test substance (step S107).

FIG. 6 is a diagram showing an example of drug efficacy evaluation according to this embodiment. As shown in FIG. 6(a), changes in average circularity were determined for two series of samples independently prepared in a dilution series in which the concentration of the test substance was lowered by half. As shown in the figure, there was a clear correlation between the concentration of the test substance and the average circularity value, and the higher the concentration, the higher the average circularity. Furthermore, there was no large difference in the numerical values between the two series, indicating that they were reproducible.

The graph shown in FIG. 6(b) shows the change in average circularity as the concentration of the test substance changes. The horizontal axis is represented by sample numbers assigned in descending order of the concentration of the test substance in this dilution series. Note that sample number 8 is a sample that is not inoculated with the test substance. If you look at the graph obtained by taking the average of the two series, you will notice that the line for sample numbers 1 to 4 is different from the line for sample numbers 4 to 8. There is not much difference in circularity between sample numbers 4 and 5 immediately after the slope changes significantly. From this, the threshold value for determining the presence or absence of medicinal efficacy can be set within this range (0.0063 to 0.0031 U/ml).

Other evaluation methods, specifically, results determined by careful visual observation multiple times by multiple experts, indicate that the test substance has a medicinal effect and is determined to be positive. The concentration of was 0.0031 U/ml or more. This is in good agreement with the above results. For these reasons, the above method can be used to determine whether or not the test substance has a medicinal effect, even for unknown samples such as samples inoculated with a test substance at any concentration or samples obtained by culturing specimens collected from subjects. be able to. Specifically, for example, it can be done as follows.

FIG. 7 is a flowchart showing a method for evaluating drug efficacy for unknown samples. The basic processing is the same as that shown in FIG. 1, and the common processing contents will be given common step numbers and the explanation will be omitted. In this process, it is assumed that a threshold value has been set in advance for the value of average circularity through prior experiments. In the case described above, for example, the average circularity value (approximately 0.3) corresponding to sample number 5 can be set as the threshold value.

First, a bright field image of an unknown sample to be evaluated is acquired as an original image (step S401). Unknown samples include samples that have been inoculated with a test substance at an unknown concentration, or samples that have been cultured from specimens collected from a subject and may have been exposed to the same chemical substance as the test substance. Can be used. By performing steps S102 to S106 on this sample in the same manner as the above processing, the average circularity of the cell colony in the image of the unknown sample is determined. Based on the comparison between this average circularity value and a threshold value, it is possible to determine the presence or absence of medicinal efficacy. That is, when the calculated average circularity is higher than the threshold value, it can be determined that there is a medicinal effect (positive).

In this way, it is possible to objectively obtain the same judgment result as an expert in a shorter time without requiring the trouble of judgment by an expert and without being influenced by his/her biases. For example, by using this technology to determine whether a specimen collected from a patient is affected by a pathogen or the toxin it produces, it can be determined whether the patient is infected with the pathogen or the toxin-producing pathogen. This makes it possible to efficiently diagnose.

FIG. 8 is a diagram illustrating the configuration of the entity that executes the drug efficacy evaluation process of this embodiment. FIG. 8(a) is an example of a configuration using a general computer device that does not have an imaging function, and FIG. 8(b) is an example of a configuration in which an imaging function is further added. Any of these can function as an execution entity of the drug efficacy evaluation process of this embodiment.

A computer device 1a shown in FIG. 8A has a general configuration as, for example, a personal computer, and includes a CPU (Central Processing Unit) 10, a memory 14, a storage 15, an input device 16, a display section 17, and an interface 18. and a disk drive 19.

By executing a control program prepared in advance, the CPU 10 implements the image processing unit 11 as a functional block for executing the above-described drug efficacy evaluation process in terms of software. Note that dedicated hardware for realizing the image processing section 11 may be provided. The memory 14 temporarily stores various data generated during the calculation process of the CPU 10. The storage 15 stores, in addition to the control program to be executed by the CPU 10, image data of original images, processed image data, etc. for a long period of time.

The input device 16 is for receiving instruction input from an operator, and includes, for example, a mouse, a keyboard, and the like. The display unit 17 is, for example, a liquid crystal display having a function of displaying images, and displays various information such as original images, processed images, and messages to the operator. Note that a touch panel in which an input device and a display unit are integrated may be provided.

The interface 18 exchanges various data with an external device via a telecommunications line. The disk drive 19 accepts an external recording disk 2 on which various data such as image data and control programs are recorded. Image data, control programs, etc. stored on the recording disk D are read out by the disk drive 19 and stored in the storage 16. The disk drive 19 may have a function of writing data generated within the computer device 1 onto the recording disk D.

The control program for causing the computer device 1 to execute the image processing of this embodiment may be read out by the disk drive 19 accessing the recording disk D on which it is recorded, and may be read out via the interface 18. It may also be an aspect provided by the device.

The same applies to original image data corresponding to an original image obtained by imaging a sample. Devices for taking bright-field images of samples and converting them into data have already been put into practical use, and the computer device 1a can receive the original image data taken by these imaging devices and execute the above-described drug efficacy evaluation process. . The original image data can be received via a recording medium such as the disk D, or can also be received through communication with an external device via the interface 18. In this case, the process of "obtaining an original image" is a process of obtaining original image data captured by another device via a recording medium or a telecommunications line. In this way, the drug efficacy evaluation process of this embodiment can be executed by the computer device 1a, which itself does not have an imaging function.

The imaging system 1b shown in FIG. 8(b) has a configuration in which an imaging section 12 is added to the configuration of the computer device 1a described above. This imaging system 1b may be configured such that a computer device having the same configuration as the computer device 1a controls an imaging section 12 configured separately from the computer device 1a, or may control the imaging section 12 and the imaging section 12. The CPU 10 and the like may be integrated. In this case, the process of "obtaining an original image" is a process in which the CPU 10 controls the imaging unit 12 to capture a bright field image of the sample, thereby obtaining the original image. In this way, the drug efficacy evaluation process of this embodiment can also be executed by being implemented in the imaging system 1b as part of its functions.

Note that the present invention is not limited to the embodiments described above, and various changes other than those described above can be made without departing from the spirit thereof. For example, in the embodiment described above, the idea of the present invention is applied to a toxicity test of a test substance (specifically, pertussis toxin produced by Bordetella pertussis) against specific cells. is not limited to the above and is arbitrary.

Further, in the drug efficacy evaluation process (FIG. 1) of the above embodiment, narrowing down of target objects (step S104) is performed after the visual field selection process (step S103), but the order may be reversed. That is, in the embodiment described above, visual field selection is performed while including cell colonies that are not suitable for shape evaluation, and target objects are narrowed down for the visual field selected as the evaluation target area. However, prior to visual field selection, objects unsuitable for processing may be excluded.

For example, in the process of the embodiment described above, after object extraction is performed on the entire original image (step S102), division into block images (step S301) is executed. However, it is possible to obtain similar results even if this order is changed and objects are extracted within the divided block image.

Further, for example, in the visual field selection process (FIG. 3), the order in which each parameter serving as a selection condition is determined is arbitrary. In other words, in this embodiment, the side lengths of the Delaunay triangle and their standard deviations are calculated in this order, and the confluency, that is, the area ratio of the cell colony in the block image, is calculated in this order, but these are independently calculated. can be calculated, and the order may be reversed.

For example, in the visual field selection process (step S103) of the above embodiment, the length of the side of the Delaunay triangle, its standard deviation, and confluency are used as parameters as selection conditions, but some of these are used for the determination. It may be an aspect. Alternatively, other indicators indicating suitability as an evaluation target area may be added to the selection conditions. Furthermore, it is also possible to omit the visual field selection process itself by setting the entire original image as the evaluation target area.

Furthermore, for example, the "appropriate range" set for each parameter in the visual field selection process may be changed depending on the purpose of evaluation. For example, in a toxicity test that evaluates the toxicity of a test substance to cells as in the above embodiment, if a toxic effect is observed in some areas of the sample, it must be determined as "toxic". be. In this sense, it is desirable that a region of the original image where the influence of toxicity is most prominent is selected as the evaluation target region. On the other hand, when the purpose is to determine the concentration of a test substance required to obtain a desired drug effect, selecting a region where an extreme drug effect appears may lead to misjudgments. If the criteria for selecting the evaluation target area in the visual field selection process can be changed, it will be possible to meet these various purposes.

As described above by exemplifying specific embodiments, in the drug efficacy evaluation method according to the present invention, for example, the circularity of an object is determined by dividing the area of the object by the square of the circumference. It can be calculated based on This definition of circularity is generally recognized, and it has been confirmed through experiments by the inventor of the present invention that good results can be obtained by using this definition in the present invention.

Furthermore, for example, the efficacy of the test substance can be determined based on a comparison between the average circularity value and a threshold value set for the average value. According to such a configuration, qualitative knowledge that the shape of cells changes depending on the efficacy of the test substance can be reflected in the quantitative evaluation. In this case, for example, a concentration value at which the rate of change in the average value of circularity with respect to a change in the concentration of the test substance changes significantly can be set as the threshold value. The threshold value set in this way has critical significance regarding the influence of the test substance on cells. Furthermore, by utilizing this fact, the average value of circularity obtained for the bright field image of the unknown sample in which the amount of the test substance is unknown is compared with the threshold value, and the presence or absence of the medicinal effect of the test substance in the unknown sample is determined. It is also possible.

For example, the evaluation target area may include a step of setting an evaluation target area prior to calculating the circularity, and the evaluation target area is a partial area of the original image, and the area occupied by the object in the area and the distance between the objects are It may be an area that satisfies predetermined conditions. In order to accurately perform quantitative evaluation based on the shape of cells or cell colonies, cells or cell colonies of appropriate size must be distributed isolated from each other at an appropriate density within the evaluation target area. desirable. By setting appropriate conditions for the area occupied by objects and the distance between objects, it becomes possible to select an area according to the purpose as an evaluation target area.

Specifically, for example, the condition regarding the area may be that the ratio of the total area of the object to the area of the area is within a predetermined range. In addition, the conditions for distance are that the distance between two objects is determined as the distance between the centers of gravity of both objects, and the value of the standard deviation of the population is the distance determined between each pair of adjacent objects. may be within a predetermined range.

A method of selecting a pair of objects that are adjacent to each other is, for example, when the original image is divided into Voronoi using the center of gravity of the object as the generating point, two objects that correspond to the generating points of two Voronoi regions that touch each other are determined to be adjacent objects. There is a method called . By performing Voronoi division, which is a well-known image process, the center of gravity of each object is divided into different regions. According to the principle of Voronoi partitioning, regions that share a boundary can be said to be "adjacent".

Based on a similar idea, the distance between a pair of adjacent objects can be determined as the length of the side when the original image is divided into Delaunay triangulations based on the centroids of the objects. When performing Delaunay triangulation, which is a well-known image processing method, on an image in which the centroids of each object are distributed, each side of the triangle is generated to connect the centroids of adjacent objects, and the length of the side is determined by the distance between the centroids. It will be expressed. Therefore, it becomes possible to identify adjacent objects and even determine the distance between their centroids.

Furthermore, for example, objects whose areas are within a predetermined range among the objects extracted within the evaluation target region may be subject to calculation of the average value of circularity. In particular, in quantitative evaluation based on the shape of cell colonies, objects that correspond to colonies from a small number of cells where the shape of each individual cell is strongly reflected in the shape of the colony, objects that correspond to a state where multiple cell colonies are in contact, etc. may deviate from the original cell colony shape. If the shape of such an object is used for evaluation, it may cause a misjudgment regarding the true efficacy of the test substance. By excluding such objects from calculation targets and selecting only objects with appropriate areas as calculation targets, it is possible to suppress such misjudgments.

This invention can be applied to the purpose of investigating the effects of test substances on cultured cells, and is useful, for example, for drug discovery that works effectively in vivo, and for investigating the toxicity of specific substances on living organisms. be able to.

1a Computer device 1b Imaging system 10 CPU
11 Image processing section 12 Imaging section D Recording disk (recording medium)

Claims (12)

acquiring a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations;
For each of the original images, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
A method for evaluating drug efficacy, comprising the step of determining the efficacy of the test substance on the cells based on the aspect of the change in the average value with respect to the change in the concentration of the test substance. 2. The medicinal efficacy evaluation method according to claim 1, wherein the circularity of the object is determined based on a value obtained by dividing the area of the object by the square of the circumference. acquiring a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations;
For each of the original images, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
and determining the efficacy of the test substance on the cells based on the change in the average value with respect to the concentration of the test substance,
The average value at a concentration at which the rate of change of the average value changes significantly with respect to a change in the concentration of the test substance is set as a threshold ;
A drug efficacy evaluation method, wherein the efficacy is determined based on a comparison between the average value and the threshold value. 4. The method according to claim 3, wherein the presence or absence of a medicinal effect of the test substance in the unknown sample is determined based on a comparison between the average value obtained for a bright field image of an unknown sample in which the amount of the test substance is unknown and the threshold value. Method for evaluating drug efficacy. acquiring a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations;
For each of the original images, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
determining the efficacy of the test substance on the cells based on the change in the average value with respect to the concentration of the test substance;
a step of setting the evaluation target area prior to calculating the circularity,
The evaluation target area is a partial area of the original image, and is an area where an area occupied by the object in the area and a distance between the objects satisfy predetermined conditions. The condition for the area is that the ratio of the total area of the object to the area of the area is within a predetermined range;
The condition for the distance is that the distance between the two objects is determined as the distance between the centroids of both objects, and the standard deviation is set to the population of the distances determined between the pairs of adjacent objects. or the value of the variance is within a predetermined range;
The method for evaluating drug efficacy according to claim 5. The drug efficacy evaluation method according to claim 6, wherein when the original image is divided into Voronoi segments using the center of gravity of the object as a generating point, the two objects corresponding to the generating points of two Voronoi regions that touch each other are the adjacent objects. . 8. The drug efficacy evaluation method according to claim 6, wherein the distance between the objects in the pair of adjacent objects is determined as the length of a side when the original image is divided into Delaunay triangulation based on the center of gravity of the objects. acquiring a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations;
For each of the original images, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
and determining the efficacy of the test substance on the cells based on the change in the average value with respect to the concentration of the test substance,
A medicinal efficacy evaluation method, wherein among the objects extracted within the evaluation target region, objects whose areas are within a predetermined range are subject to calculation of the average value of circularity. For each of a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
A computer program for causing a computer to execute a step of determining the efficacy of the test substance on the cells based on a change in the average value with respect to a change in the concentration of the test substance. For each of a plurality of original images obtained by bright-field imaging each of a plurality of samples obtained by inoculating or administering a test substance to cultured cells at different concentrations, extracting an object corresponding to the cell in the original image;
For each of the original images, calculating the circularity of each of the objects included in the evaluation target area in the original image and finding an average value thereof;
and determining the efficacy of the test substance on the cells based on the change in the average value with respect to the concentration of the test substance,
The average value at a concentration where the rate of change of the average value changes significantly with respect to a change in the concentration of the test substance is set as a threshold value, and the process of determining the efficacy based on a comparison between the average value and the threshold value is performed by a computer. A computer program for running. A computer-readable recording medium on which the computer program according to claim 10 or 11 is recorded.

Images