Patent Application
20250029407
The technology of the present disclosure relates to an evaluation system, an information processing device, and an information processing method.
In order to improve an efficiency of new drug development, for example, a method of performing a toxicity evaluation using cells such as myocardial cells produced from induced pluripotent stem (iPS) cells has been developed (see, for example, WO2019/131806A). The toxicity evaluation is performed by evaluating responsiveness of cells to a drug.
As a culture vessel for the cells, for example, a well plate in which a plurality of wells are formed is used. A microelectrode array (MEA) in which a plurality of minute measurement electrodes are disposed is provided on a bottom surface of each well. Such a well plate is called an MEA plate. A waveform (for example, a myocardial waveform indicating pulsation of the myocardial cells) indicating an electrophysiological change of cells cultured in the wells is output from each measurement electrode of the microelectrode array. The toxicity evaluation is performed by measuring a change in waveform with respect to a drug.
Variations occur in the waveform output from each of the plurality of measurement electrodes provided in the wells. Therefore, for each well, one measurement electrode from which the most ideal waveform is output is selected as a target electrode to be a target for the toxicity evaluation. Such selection of the target electrode is also called selection of the golden channel. For example, in a case of the myocardial cells, a waveform closest to the healthiest state is selected among the myocardial waveforms output from the measurement electrodes based on a known myocardial waveform representing a healthy state (for example, a state without a disease such as arrhythmia) obtained in the past measurement.
However, the measurement electrode (that is, the golden channel) selected as the target electrode is merely an electrode that is relatively suitable for the toxicity evaluation among a plurality of measurement electrodes whose waveforms are measured. That is, this does not guarantee that a waveform output from the golden channel is truly measured correctly. For example, in a case in which the cells are not appropriately seeded in the well, the waveform is not suitable for the toxicity evaluation, resulting in reduced evaluation accuracy.
An object of the technology of the present disclosure is to provide an evaluation system, an information processing device, and an information processing method that enable improvement in evaluation accuracy of a toxicity evaluation.
In order to achieve the above object, an evaluation system of the present disclosure is an evaluation system that evaluates a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the evaluation system comprising: a microscope device that generates image data by imaging the electrode and the cells from a surface side of the electrode of the cell culture vessel; and an information processing device that analyzes the image data and determines whether the seeding state is appropriate based on an analysis result.
It is preferable that the microscope device is an epifluorescence microscope that generates the image data by irradiating the cells stained with a fluorescent dye with excitation light and capturing an image of fluorescence emitted from the cells.
It is preferable that the microscope device generates the image data for specifying a region of the cells and a region of the electrode by capturing an image of the excitation light reflected by the electrode in addition to the fluorescence.
It is preferable that the microscope device generates the image data for specifying a region of the cells and a region of the electrode by irradiating the cells and the electrode with illumination light and capturing an image of reflected light from the cells and the electrode.
It is preferable that the electrode includes a measurement electrode for measuring a potential and at least one of a reference electrode to which a reference potential is applied or a stimulation electrode for applying a stimulus to the cells.
It is preferable that the cells are smaller than a size of the measurement electrode, and a plurality of the cells are seeded on the measurement electrode.
It is preferable that the plurality of seeded cells exhibit a single behavior.
It is preferable that the cells are myocardial cells.
It is preferable that the information processing device calculates a cell coverage on the reference electrode or on the stimulation electrode by analyzing the image data, and determines that the seeding state is appropriate in a case in which the calculated cell coverage is equal to or less than a threshold value.
It is preferable that in a case in which the cell coverage is not equal to or less than the threshold value, the information processing device estimates an influence on a measurement waveform based on the cell coverage.
It is preferable that the information processing device analyzes a state of the cells on the measurement electrode based on the image data.
It is preferable that the information processing device issues a warning in a case in which the seeding state is not appropriate.
An information processing device of the present disclosure is an information processing device that evaluates a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the information processing device comprising: a processor, in which the processor acquires image data generated by imaging the electrode and the cells from a surface side of the electrode of the cell culture vessel with a microscope device, and analyzes the image data and determines whether the seeding state is appropriate based on an analysis result.
An information processing method of the present disclosure is an information processing method of evaluating a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the information processing method comprising: acquiring image data generated by imaging the electrode and the cells from a surface side of the electrode of the cell culture vessel with a microscope device; and analyzing the image data and determining whether the seeding state is appropriate based on an analysis result.
According to the technology of the present disclosure, it is possible to provide an evaluation system, an information processing device, and an information processing method that enable improvement in evaluation accuracy of a toxicity evaluation.
Exemplary embodiments according to the technique of the present disclosure will be described in detail based on the following figures, wherein:
Hereinafter, an embodiment according to the technology of the present disclosure will be described with reference to the drawings.
An MEA plate 40 is used for culturing the cells. The potential measurement device 10 is provided with an accommodation part 11 that accommodates the MEA plate 40 therein. In addition, the potential measurement device 10 is provided with a slide-type lid 12 for opening and closing the accommodation part 11. The MEA plate 40 is accommodated in the accommodation part 11 with the cells seeded thereon.
In the present embodiment, myocardial cells produced from iPS cells are used as the cells. In addition, the potential measurement device 10 measures an extracellular potential representing a myocardial waveform of the myocardial cells seeded on the MEA plate 40 by a multipoint measurement method, and outputs waveform data obtained by the measurement to the information processing device 30. The waveform data represents a pulse signal output by the myocardial cells.
The microscope device 20 generates image data by imaging a plurality of culture wells (hereinafter, simply referred to as wells) 42 formed in the MEA plate 40, on which the waveform is measured by the potential measurement device 10, and outputs the generated image data to the information processing device 30.
The information processing device 30 is configured of a general computer such as a personal computer. The information processing device 30 is installed with software for analyzing the waveform data input from the potential measurement device 10 and for analyzing the image data input from the microscope device 20.
The information processing device 30 includes a display unit 31 and an input unit 32. The display unit 31 is a display device such as a liquid crystal display or an organic electro luminescence (EL) display. The input unit 32 is an input device such as a keyboard, a touch pad, or a mouse. The information processing device 30 is connected to the potential measurement device 10 and the microscope device 20 via a wired or wireless connection. The display unit 31 and the input unit 32 may be configured as external devices connected to the information processing device 30.
The information processing device 30 calculates an extracellular potential duration (field potential duration (FPD)), an interspike interval (ISI), and the like based on the input waveform data. Since the FPD corresponds to a QT interval (time from a start of a Q wave to an end of a T wave) in an electrocardiogram, it is used as an index of arrhythmia. Prolongation of the QT interval indicates a potential for arrhythmia. It is possible to perform a toxicity evaluation for evaluating responsiveness of the cells to a drug candidate compound, based on the FPD or the like.
In addition, the information processing device 30 analyzes the image data input from the microscope device 20 and determines whether a seeding state of the cells is appropriate based on an analysis result.
In addition to the microelectrode array 50, a reference electrode 53 is provided on the bottom surface 44 of the well 42. The reference electrode 53 is disposed around the microelectrode array 50 along an outer edge of the bottom surface 44 so as not to come into contact with the cells to be measured. A ground potential as a reference potential is applied to the reference electrode 53. The extracellular potential is measured from a potential difference between the measurement electrode 51 and the reference electrode 53. One cell to be seeded is smaller than a size of the measurement electrode 51. A plurality of cells (approximately 2 to 3 cells) are seeded on one measurement electrode 51.
In addition, a stimulation electrode 54 for applying a stimulus to the cells is provided on the bottom surface 44 of the well 42. Note that it is not essential to apply a stimulus to the cells, and the stimulation electrode 54 does not have to be provided in the well 42.
The information processing device 30 includes a processor 33, a storage unit 34, the input unit 32, the display unit 31, a communication I/F 35, and a bus 36. The processor 33 is a computer that realizes various functions by reading out a program 38 and various kinds of data stored in the storage unit 34 and executing processing. The processor 33 is, for example, a central processing unit (CPU).
The storage unit 34 is a storage device that stores the program 38 and the various kinds of data in a case in which the processor 33 executes processing. The storage unit 34 includes, for example, a random access memory (RAM), a read only memory (ROM), or a storage. The RAM is, for example, a volatile memory used as a work area or the like of the processor 33. The ROM is, for example, a non-volatile memory such as a flash memory that holds the program 38 and various kinds of data. The storage is, for example, a large-capacity storage device such as a hard disk drive (HDD) or a solid state drive (SSD), and stores an operating system (OS), various kinds of data, and the like. The storage unit 34 may be configured as an external apparatus connected to the information processing device 30.
Variations occur in the waveform output from the plurality of measurement electrodes 51 included in the microelectrode array 50. For the toxicity evaluation, it is necessary to use a waveform close to an ideal waveform. The ideal waveform is, for example, a waveform representing a healthy state (for example, a state without a disease such as arrhythmia) obtained by the past measurement. From among the plurality of measurement electrodes 51, the measurement electrode 51 that outputs a waveform with the highest degree of similarity to the ideal waveform is selected as the target electrode for the toxicity evaluation. Such selection of the target electrode is also referred to as selection of a golden channel (hereinafter, referred to as GC).
The toxicity evaluation is performed by evaluating a change in waveform (for example, an extension of a QT interval) caused by adding a drug candidate compound to the culture solution of the well 42.
The information processing device 30 evaluates the degree of similarity to the ideal waveform using the FPD, the ISI, the maximum potential of P1, the minimum potential of P1, the value of A2, and the overall shape of the waveform as evaluation criteria.
The microscope device 20 is an epi-illumination microscope that images the measurement electrode 51 and the cells from a surface side of the measurement electrode 51 to which the cells adheres. A transmission microscope that captures an image of transmitted light of light applied to a back surface side of the measurement electrode 51 cannot image the cells on the measurement electrode 51, so that, in the present embodiment, the microscope device 20 is used as an epi-illumination microscope.
The microscope device 20 is an epifluorescence microscope configured to include a light source 21, an excitation filter 22, a dichroic mirror 23, an objective lens 24, an absorption filter 25, and an imaging sensor 26. The light source 21 is, for example, a mercury lamp, and emits light L having wavelengths from ultraviolet to near-infrared. The excitation filter 22 is a bandpass filter that extracts light having a wavelength required for excitation of a fluorescent substance (hereinafter, referred to as excitation light EL) from the light L emitted from the light source 21.
The dichroic mirror 23 reflects the excitation light EL and transmits fluorescence FL. The excitation light EL reflected by the dichroic mirror 23 passes through the objective lens 24 and is emitted from the opening 43 toward the bottom surface 44 of the well 42 to which the stained cell group is attached. The cell group is irradiated with the excitation light EL, thereby emitting the fluorescence FL. The fluorescence FL emitted from the cell group is transmitted through the objective lens 24 and the dichroic mirror 23 and is incident into the absorption filter 25.
The absorption filter 25 transmits the fluorescence FL and absorbs unnecessary scattered light other than the fluorescence FL. The imaging sensor 26 captures an image of the fluorescence FL transmitted through the absorption filter 25 to generate and output image data.
In the present embodiment, in order to image the electrodes including the reference electrode 53 and the measurement electrode 51 in addition to the cell group, the dichroic mirror 23 and the absorption filter 25 are configured to transmit a part of the excitation light EL reflected by the electrodes. That is, a region of the cells and a region of the electrodes on the bottom surface 44 of the well 42 can be specified based on the image data output from the imaging sensor 26.
In addition, the microscope device 20 is provided with a moving mechanism (not shown) that sequentially changes the wells 42 to be imaged, and the wells 42 in the MEA plate 40 are sequentially imaged.
An image 71 shown in
Even in a case in which the measurement electrode 51 from which the waveform shown in
In addition, in a case in which cells are present on the reference electrode 53 to which the ground potential is applied as the reference potential as described above, the reference potential changes, an offset occurs in the measurement waveform as described above.
In a case in which the cells are myocardial cells, the cell group synchronously exhibits a single behavior and acts, so that, in a case in which the waveform measurement is performed by the measurement electrode 51, a change in the reference potential inevitably occurs in the reference electrode 53 at the same time. Therefore, the influence on the measurement waveform due to the presence of the cells on the reference electrode 53 is large. On the other hand, in a case in which the cells are cells that act asynchronously like a nerve cell, the reference electrode 53 having a considerably large area is used, so that the potential change on the reference electrode 53 is averaged. Therefore, the influence on the measurement waveform due to the presence of the cells on the reference electrode 53 is smaller than in a case in which the cells are myocardial cells.
The processor 33 functions as a waveform data acquisition unit 80, a waveform analysis unit 81, an output unit 82, an image data acquisition unit 83, an image analysis unit 84, and a determination unit 85. The waveform data acquisition unit 80 acquires a plurality of the waveform data output from the potential measurement device 10. The output unit 82 displays the waveform, the analysis result of the waveform data, and the like on the display unit 31.
The image data acquisition unit 83 acquires the image data from the microscope device 20. The image analysis unit 84 calculates the above-described cell coverage based on the image data. The determination unit 85 determines whether the seeding state of the cells is appropriate based on the cell coverage calculated by the image analysis unit 84. The output unit 82 displays the determination result of the seeding state determined by the determination unit 85 on the display unit 31. For example, the output unit 82 displays the determination result of the seeding state for each well 42 on the display unit 31.
In a case in which the determination unit 85 determines that the seeding state of the cells is inappropriate, the output unit 82 may issue a warning to a user by displaying a message or the like on the display unit 31.
In a case in which the cell coverage is not equal to or less than the threshold value, the determination unit 85 may perform a process of estimating the influence (for example, the offset amount of the potential during the extracellular potential duration) on the measurement waveform based on the cell coverage. In this case, the output unit 82 displays the estimated value estimated by the determination unit 85, on the display unit 31. The user can determine whether the waveform is acceptable for the toxicity evaluation based on the estimated value.
Next, the MEA plate 40 is taken out from the potential measurement device 10, and the cell group is stained (step S23). After the cell group is stained, a plurality of image data are acquired by sequentially imaging the wells 42 of the MEA plate 40 using the microscope device 20 (step S24). The plurality of image data acquired by the microscope device 20 are output to the information processing device 30, and image analysis is performed on each piece of image data by the information processing device 30 (step S25). In step S25, the calculation of the cell coverage is performed. Then, the information processing device 30 determines the seeding state of the cells based on the calculated cell coverage (step S26), and displays the determination result on the display unit 31 (step S27).
As described above, according to the technology of the present disclosure, the reliability of the waveform (that is, whether the waveform is obtained by appropriate measurement) can be grasped based on the determination result of the seeding state, and the toxicity evaluation can be performed using the waveform with high reliability, thereby improving the evaluation accuracy of the toxicity evaluation.
Hereinafter, various modification examples of the above-described embodiment will be described.
In the above-described embodiment, the image analysis unit 84 calculates the cell coverage of the reference electrode 53, but in a case in which the cell group is stimulated, a cell coverage of the stimulation electrode 54 may be calculated instead of or in addition to the reference electrode 53. This is because, in a case in which the stimulation electrode 54 is covered with cells, the stimulus applied from the stimulation electrode 54 to the cells is reduced below a desired intensity, which may affect the waveform.
In addition, in the above-described embodiment, the seeding state of the cells is determined based on the cell coverage of the reference electrode 53, but it may be determined whether the seeding state is appropriate based on other factors that are considered to affect the measurement waveform. For example, it may be determined whether the seeding state is appropriate by analyzing a state of the cells on the measurement electrode 51, such as the adhesiveness between the measurement electrode 51 and the cells, the cell density on the measurement electrode 51, and the variation in the cells on the measurement electrode 51.
The adhesiveness between the measurement electrode 51 and the cells can be evaluated by obtaining a thickness of the cells, a flatness of the cells, a distance between the measurement electrode 51 and the cells, and the like. The adhesiveness between the measurement electrode 51 and the cells can be accurately evaluated by using a confocal microscope, which is a type of epifluorescence microscope, as the microscope device 20. The confocal microscope can generate a three-dimensional image by acquiring images from a plurality of focal planes. The seeding state is more appropriate as the adhesiveness between the measurement electrode 51 and the cells increases. The adhesiveness between the measurement electrode 51 and the cells can also be determined by imaging the cells stained with a fluorescent dye for a factor related to the adhesiveness.
The cell density on the measurement electrode 51 increases as cells are laminated on the measurement electrode 51. For example, as shown in
The variation in the cells on the measurement electrode 51 affects the expression level of the ion channel. It is preferable that the variation in the cells on the measurement electrode 51 is within an appropriate range.
In addition, since the waveform changes depending on the maturity of the cells, the maturity of the cells may be analyzed from the image data. The maturity of the cells can be determined using the cell morphology or fluorescence labeling. In a case in which the fluorescence labeling is used, a factor related to the maturity of the cells need only be stained with a fluorescent dye. For example, in a case in which the maturity of the cells is high, the cell morphology becomes oriented (that is, the cell elongates in a specific direction). In this way, the maturity of the cells can be determined by the orientation.
In addition, in the above-described embodiment, the microscope device 20 is used as an epifluorescence microscope, but a reflective type microscope can also be used instead of the epifluorescence microscope. In a case in which a reflective type microscope is used, the cell morphology is imaged without staining the cells with a fluorescent dye. In a case in which a reflective type microscope is used as the microscope device 20, the bottom surface 44 of the well 42 is illuminated with illumination light from obliquely upward, and an image of reflected light from the cells, the reference electrode 53, and the like is captured to generate image data. Further, instead of the epifluorescence microscope, a phase contrast microscope may be used to image the cell morphology without staining the cells with a fluorescent dye. The reflective type microscope and the phase contrast microscope can image the cell morphology without staining the cells, that is, non-destructively.
In addition, a model that is trained through machine learning may be used to acquire information equivalent to information obtained in a case in which the cells are imaged with an epifluorescence microscope, from the image data obtained by imaging the cells non-destructively using the reflective type microscope, the phase contrast microscope, or the like. For example, the model that is trained through machine learning is a deep learning model such as a convolutional neural network. The model that is trained through machine learning is a learning model that has been trained through machine learning using the image data obtained by imaging the cells non-destructively as input data and the image data obtained by imaging the cells with an epifluorescence microscope as correct answer data.
In addition, in the above-described embodiment, the imaging is performed by the microscope device 20 after the waveform measurement is performed by the potential measurement device 10, but the imaging may be performed by the microscope device 20 before the waveform measurement is performed by the potential measurement device 10. In a case in which a reflective type microscope is used as the microscope device 20, it is not necessary to stain the cells with a fluorescent dye, so that the imaging can be performed before the waveform measurement is performed. In addition, even though an epifluorescence microscope is used as the microscope device 20, in a case in which a fluorescent dye that does not affect the waveform measurement is used, it is also possible to perform the imaging before performing the waveform measurement.
In addition, in the above-described embodiment, the potential measurement device 10 and the microscope device 20 are configured as separate devices, but the microscope device 20 may be incorporated into the potential measurement device 10.
In addition, in the above embodiment, the potential measurement device 10 and the microscope device 20 are connected to one information processing device 30, but the potential measurement device 10 and the microscope device 20 may be connected to separate information processing devices.
In addition, in the above embodiment, the evaluation system 2 is configured to include the potential measurement device 10, the microscope device 20, and the information processing device 30, but the evaluation system according to the technology of the present disclosure need only include the microscope device and the information processing device.
In the above-described embodiment, the myocardial cells are used as the cells, but it is also possible to use cells such as nerve cells instead of the myocardial cells.
In the above-described embodiment, for example, a hardware structure of a processing unit that executes various kinds of processing, such as the waveform data acquisition unit 80, the waveform analysis unit 81, the output unit 82, the image data acquisition unit 83, the image analysis unit 84, and the determination unit 85, is various processors as shown below.
Various processors include a CPU, a programmable logic device (PLD), a dedicated electric circuit, and the like. As is well known, the CPU is a general-purpose processor that executes software (program) and functions as various processing units. The PLD is a processor whose circuit configuration can be changed after manufacturing, such as a field programmable gate array (FPGA). The dedicated electric circuit is a processor that has a dedicated circuit configuration designed to perform a specific process, such as an application specific integrated circuit (ASIC).
One processing unit may be configured of one of these various processors, or may be configured of a combination of two or more processors of the same type or different types (for example, a combination of a plurality of FPGAs or a combination of a CPU and an FPGA). In addition, a plurality of processing units may be constituted by one processor. As an example in which the plurality of processing units are constituted by one processor, first, one processor is constituted by a combination of one or more CPUs and software and this processor functions as the plurality of processing units. Second, as typified by a system on chip (SoC) or the like, a processor that realizes the functions of the entire system including the plurality of processing units by using one IC chip is used. As described above, the various processing units are configured using one or more of the various processors as a hardware structure.
More specifically, the hardware structure of these various processors is an electric circuit (circuitry) in which circuit elements such as semiconductor elements are combined.
The present disclosure is not limited to the embodiment described above and may adopt various configurations without departing from the spirit of the present disclosure. In addition to the program, the present disclosure extends to a computer-readable storage medium that stores the program in a non-temporary manner.
It is possible to grasp the following technologies by the above description.
An evaluation system that evaluates a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the evaluation system comprising:
The evaluation system according to Appendix 1,
The evaluation system according to Appendix 2,
The evaluation system according to Appendix 1,
The evaluation system according to Appendix 3 or 4,
The evaluation system according to Appendix 5,
The evaluation system according to Appendix 6,
The evaluation system according to Appendix 7,
The evaluation system according to any one of Appendices 5 to 8,
The evaluation system according to Appendix 9,
The evaluation system according to any one of Appendices 5 to 8,
The evaluation system according to any one of Appendices 1 to 11,
An information processing device that evaluates a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the information processing device comprising:
An information processing method of evaluating a seeding state of cells seeded in a cell culture vessel having at least one electrode disposed on a bottom surface, the information processing method comprising:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-063642 | Apr 2022 | JP | national |
This application is a continuation application of International Application No. PCT/JP2023/014108, filed Apr. 5, 2023, the disclosure of which is incorporated herein by reference in its entirety. Further, this application claims priority from Japanese Patent Application No. 2022-063642, filed on Apr. 6, 2022, the disclosure of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2023/014108 | Apr 2023 | WO |
| Child | 18905978 | US |
