Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/58—Reaction vessels connected in series or in parallel
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
  • C12M33/07—Dosage or metering devices therefore
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
  • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/48—Automatic or computerized control
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5058—Neurological cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5061—Muscle cells

Definitions

• the technology of the present disclosure relates to a cell seeding device, an information processing device, and an information processing method.
• Toxicity evaluation is performed by evaluating the responsiveness of cells to drugs.
• a well plate in which a plurality of wells are formed is used as a cell culture container.
• a microelectrode array (MEA) in which a plurality of microscopic measurement electrodes are arranged is provided on the bottom of each well.
• a reference electrode to which a reference potential is applied is provided on the bottom surface of each well around the microelectrode array (see, for example, Japanese Patent Laid-Open No. 2021-179319).
• Each measurement electrode of the microelectrode array outputs a waveform indicating electrophysiological changes in cells cultured in the well (for example, a myocardial waveform indicating the pulsation of myocardial cells). Toxicity evaluation is performed by measuring changes in waveforms for drugs.
• Cells are seeded onto the MEA plate by dropping the cell suspension into each well using a pipette or the like.
• the reference electrode is an electrode to which a reference potential is applied, it is preferable that no cells are placed on the reference electrode.
• seeding is performed manually, it is not easy to place cells on the measurement electrode without placing the cells on the reference electrode because the positional accuracy of the seeding is low.
• the objective of the technology of the present disclosure is to provide a cell seeding device, an information processing device, and an information processing method that enable accurate cell seeding.
• the cell seeding device of the present disclosure includes a dropping device that drops droplets of cell suspension into a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom, and a dropping device that is provided in the dropping device. , an imaging device that captures an image of the bottom surface and generates an image, detects the positions of the measurement electrode and reference electrode based on the image, and places the cell on the measurement electrode without placing the cell on the reference electrode based on the detected information. and an information processing device that determines the dropping position of the droplet.
• the information processing device determines the dropping position and the dropping amount of the droplet based on the detection information.
• the information processing device determines a dropping amount that will prevent the dropped droplet from expanding and reaching the reference electrode, based on the relationship between the dropping amount and the size of the droplet after expansion.
• the information processing device presents the user with the expected enlarged size of the droplet based on the determined droplet amount, and changes the droplet amount based on the user's operation.
• the imaging device is preferably an image sensor that captures images using visible light or a laser-based sensing device.
• the reference electrode is preferably arranged around a microelectrode array in which a plurality of measurement electrodes are arranged in a two-dimensional array.
• the cells are cardiomyocytes or nerve cells.
• the information processing device of the present disclosure is an information processing device that determines the amount of droplets of a cell suspension to be dropped by a dropping device onto a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom surface, and the information processing device includes a processor.
• the processor detects the positions of the measurement electrode and the reference electrode based on the image generated by imaging the bottom surface of the imaging device, and the processor detects the positions of the measurement electrode and the reference electrode based on the detected information, without placing cells on the reference electrode. Determine the dropping position of the droplet that allows the cells to be placed on it.
• the information processing method of the present disclosure is an information processing method that determines the amount of droplets of a cell suspension to be dropped by a dropping device into a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom surface, and the method includes: The device detects the positions of the measurement electrode and reference electrode based on the image generated by imaging the bottom surface, and based on the detected information, it is possible to place cells on the measurement electrode without placing cells on the reference electrode. Determine the dropping position of the droplet.
• FIG. 1 is a diagram schematically showing the configuration of a cell seeding device. It is a perspective view showing an example of an MEA plate. It is a figure which shows the structural example of the bottom surface of a well.
• FIG. 1 is a block diagram showing an example of the configuration of an information processing device.
• FIG. 2 is a block diagram showing an example of functions configured in a processor and a controller. It is a figure which shows an example of the dripping position determined by the dripping position determination part. It is a flow chart which shows an example of the flow of processing of a cell seeding device. It is a figure which shows an example of the bottom surface of the well where the droplet was dripped. It is a figure showing the evaluation flow of toxicity evaluation.
• FIG. 1 is a diagram schematically showing the configuration of a cell seeding device. It is a perspective view showing an example of an MEA plate. It is a figure which shows the structural example of the bottom surface of a well.
• FIG. 1 is a block diagram
• FIG. 3 is a block diagram showing functions configured in a processor and a controller according to a first modification.
• FIG. 3 is a diagram illustrating the shortest distance between a measurement electrode and a reference electrode. It is a figure which shows an example of a reference table.
• FIG. 3 is a block diagram showing functions configured in a processor and a controller according to a second modification.
• FIG. 3 is a diagram showing an example of a GUI screen. It is a flowchart which shows an example of the flow of a dripping amount change process.
• FIG. 3 is a diagram showing a plurality of assumed areas in consideration of variations. It is a figure which shows the modification of a GUI screen.
• the vertical direction is defined as the Z direction
• one direction perpendicular to the Z direction is defined as the X direction
• the direction perpendicular to the Z direction and the X direction is defined as the Y direction.
• FIG. 1 schematically shows the configuration of the cell seeding device 2.
• the cell seeding device 2 is a device that seeds cells by dropping a cell suspension into each of a plurality of culture wells (hereinafter simply referred to as wells) 12 formed on the MEA plate 10.
• wells a plurality of culture wells
• nerve cells or cardiomyocytes produced from iPS cells are used as cells.
• the MEA plate 10 After the MEA plate 10 is seeded with cells by the cell seeding device 2 and cultured by a culture device (not shown), it is attached to a potential measuring device (not shown).
• a waveform indicating electrophysiological changes in a cultured cell group for example, a myocardial waveform indicating the pulsation of myocardial cells is measured.
• the cell seeding device 2 includes a dispenser 3, an imaging device 4, a controller 5, and an information processing device 6.
• the dispenser 3 includes a housing section 20, a dripping section 21, and a moving mechanism 22.
• the dispenser 3 is an example of a "dropping device" according to the technology of the present disclosure.
• the storage unit 20 is a tank that stores a cell suspension in which cells are suspended in a liquid medium.
• a dripping part 21 is connected to the lower end of the accommodating part 20 .
• the dripping part 21 drops the cell suspension contained in the storage part 20 toward the well 12 as a droplet DL.
• the dripping portion 21 is, for example, a nozzle having a circular cross-sectional shape and extending in the Z direction.
• the droplet portion 21 ejects droplets DL in the Z direction from the nozzle opening 21A, for example, using a piezoelectric method similar to an inkjet that ejects ink particles using a piezoelectric element.
• the droplet DL contains one or more cells.
• the moving mechanism 22 is connected to the upper end of the storage section 20.
• the moving mechanism 22 moves the storage section 20 and the dripping section 21 in the X direction, the Y direction, and the Z direction. That is, the moving mechanism 22 displaces the dripping part 21 relative to the well 12.
• the imaging device 4 is connected to the storage section 20 and moves integrally with the storage section 20 and the dripping section 21.
• the imaging device 4 images the bottom surface 14 of the well 12 onto which the droplet DL is dropped (that is, where the cells are seeded) to generate an image.
• the imaging device 4 is an image sensor that captures images using visible light. Note that a light source that irradiates illumination light onto the bottom surface 14 of the well 12 may be provided.
• the imaging device 4 may be of a reflection type or a transmission type.
• the controller 5 performs dripping control to drop droplets DL by driving the piezoelectric element of the dripping part 21, movement control to move the dropping part 21 by driving the moving mechanism 22, and instructing the imaging device 4 to perform an imaging operation. Performs imaging control to be performed.
• the information processing device 6 is configured by a general computer such as a personal computer.
• the information processing device 6 detects the electrode position within the well 12 based on the image acquired by the imaging device 4 and determines the dropping position.
• the information processing device 6 instructs the controller 5 to drop the droplet DL at the determined dropping position.
• FIG. 2 shows an example of the MEA plate 10.
• the MEA plate 10 is a multi-well plate in which a plurality of wells 12 are arranged on a substrate 11.
• the MEA plate 10 shown in FIG. 2 has 48 wells 12. Note that the number of wells 12 provided in the MEA plate 10 is not limited to 48, but may be 24, 96, etc.
• the well 12 is a substantially cylindrical container with an opening 13 (see FIG. 1) formed at the top.
• the well 12 is an example of a "cell culture device" according to the technology of the present disclosure.
• FIG. 3 shows an example of the configuration of the bottom surface 14 of the well 12.
• a microelectrode array 30 is embedded in the bottom surface 14 of the well 12 .
• the microelectrode array 30 has a plurality of measurement electrodes 31 arranged in a two-dimensional array. In the example shown in FIG. 3, the microelectrode array 30 has 16 measurement electrodes 31 arranged in a 4 ⁇ 4 square arrangement.
• the measurement electrode 31 is exposed on the bottom surface 14 of the well 12 and comes into contact with the seeded cells.
• the measurement electrode 31 has a diameter of several tens of micrometers.
• Each of the measurement electrodes 31 is connected to a potential measurement circuit of a potential measurement device via a wiring 32.
• a reference electrode 33 is provided on the bottom surface 14 of the well 12.
• the reference electrode 33 is arranged around the microelectrode array 30 along the outer edge of the bottom surface 14 so as not to come into contact with cells to be measured.
• a ground potential as a reference potential is applied to the reference electrode 33.
• the extracellular potential is measured from the potential difference between the measurement electrode 31 and the reference electrode 33.
• One seeded cell is smaller than the size of the measurement electrode 31.
• a plurality of cells (about 2 to 3 cells) are seeded on one measurement electrode 31.
• a stimulation electrode 34 for stimulating cells is provided on the bottom surface 14 of the well 12. However, it is not essential to stimulate the cells, and the stimulation electrode 34 may not be provided in the well 12.
• FIG. 4 shows an example of the configuration of the information processing device 6.
• the information processing device 6 includes a processor 40 , a storage section 41 , an input section 42 , a display section 43 , a communication I/F 44 , and a bus 45 .
• the processor 40 is a computer that implements various functions by reading out a program 46 and various data stored in the storage unit 41 and executing processes.
• the processor 40 is, for example, a CPU (Central Processing Unit).
• the storage unit 41 is a storage device that stores a program 46 and various data used when the processor 40 executes processing.
• the storage unit 41 includes, for example, RAM (Random Access Memory), ROM (Read Only Memory), or storage.
• the RAM is, for example, a volatile memory used as a work area for the processor 40.
• the ROM is, for example, a nonvolatile memory such as a flash memory that holds the program 46 and various data.
• the storage is, for example, a large-capacity storage device such as an HDD (Hard Disk Drive) or an SSD (Solid State Drive), and stores an OS (Operating System), various data, and the like.
• the storage unit 41 may be configured as an external device connected to the information processing device 6.
• the input unit 42 is an input device such as a keyboard, touch pad, or mouse.
• the display unit 43 is a display device such as a liquid crystal display.
• the input unit 42 and the display unit 43 may be configured as external devices connected to the information processing device 6.
• the processor 40 communicates with the controller 5 via the communication I/F 44.
• FIG. 5 shows an example of functions configured in the processor 40 and controller 5.
• the controller 5 includes a drip control section 5A that performs the above-described drip control, a movement control section 5B that performs the above-described movement control, and an imaging control section 5C that performs the above-described imaging control. These may be configured by either hardware or software. Furthermore, the controller 5 may be incorporated into the information processing device 6.
• the processor 40 functions as a main control section 50, an image acquisition section 51, an electrode position detection section 52, and a dropping position determination section 53. These functions are realized by the processor 40 executing processing based on the program 46 (see FIG. 4). These functions may be realized by hardware.
• the main control unit 50 comprehensively controls various functions within the controller 5 and processor 40.
• the main control unit 50 also receives operation signals input from the input unit 42 and performs display control to display various information on the display unit 43.
• the main control unit 50 instructs the imaging control unit 5C to cause the imaging device 4 to image the bottom surface 14 of the well 12.
• the image acquisition unit 51 acquires an image generated by the imaging device 4.
• the electrode position detection unit 52 detects the positions of the plurality of measurement electrodes 31 and reference electrodes 33 based on the image acquired by the image acquisition unit 51, and inputs the detection information to the dropping position determination unit 53. Since the positions of the plurality of measurement electrodes 31 and reference electrodes 33 vary from well to well 12, accurate positions are detected by the electrode position detection section 52.
• the dropping position determining unit 53 determines the dropping position of the droplet DL based on the detection information input from the electrode position detecting unit 52. Specifically, the dropping position determination unit 53 determines a position that allows cells to be placed on the measurement electrode 31 without placing the cells on the reference electrode 33 as the dropping position. If a cell is placed on the reference electrode 33 to which a reference potential is applied, the waveform measured by the potential measuring device may be affected by an offset or the like. Determine the dripping position so that it does not overlap.
• the main controller 50 places the dripping part 21 above the dripping position by giving an instruction to the movement control part 5B based on the dripping position determined by the dripping position determination part 53, and then disposes the dripping part 21 above the dripping position.
• the droplet DL is dropped at the dropping position by giving an instruction to the droplet DL.
• the electrode position detection unit 52 may detect the position of the stimulation electrode 34 in addition to the positions of the plurality of measurement electrodes 31 and reference electrodes 33.
• the dropping position determination unit 53 determines a position that allows cells to be placed on the measurement electrode 31 without placing cells on the reference electrode 33 and stimulation electrode 34 as the dropping position. This is because if cells are placed on the stimulation electrode 34, the stimulation applied to the cells from the stimulation electrode 34 will not have the desired intensity, which may affect the waveform measured by the potential measurement device.
• FIG. 6 shows an example of the dropping position determined by the dropping position determination unit 53.
• the dropping position determination unit 53 determines a dropping position DP on each measurement electrode 31 so that no cells are placed on the reference electrode 33, based on the detection information of the electrode position.
• FIG. 7 shows an example of the process flow of the cell seeding device 2. Note that FIG. 7 shows the flow of processing when cells are seeded into one well 12.
• the main control unit 50 When the main control unit 50 receives a start instruction from the input unit 42 with the MEA plate 10 set in the cell seeding device 2, it gives an instruction to the imaging control unit 5C to cause the imaging device 4 to is imaged (step S10). Images generated by the imaging device 4 are transmitted to the information processing device 6 via the controller 5.
• the image transmitted from the imaging device 4 is acquired by the image acquisition unit 51, and the electrode position is detected by the electrode position detection unit 52 based on the image acquired by the image acquisition unit 51 (step S11). .
• the dropping position DP is determined by the dropping position determining unit 53 based on the detection information of the electrode position detected by the electrode position detecting unit 52 (step S12).
• a plurality of dropping positions DP are determined by the dropping position determination unit 53.
• the main control unit 50 instructs the movement control unit 5B to move the dripping unit 21 so that the dripping unit 21 is positioned above one of the plurality of dripping positions DP (step S13).
• the main controller 50 then instructs the droplet controller 5A to drop the droplet DL (step S14).
• the main control unit 50 determines whether the drop position DP at which the drop was applied in step S14 is the final drop position (that is, whether or not the drop has been applied to all of the plurality of drop positions DP determined by the drop position determination unit 53). is determined (step S15).
• step S15 NO
• the process returns to step S13 and moves the dropping part 21 above the next dropping position DP.
• Steps S13 and S14 are repeatedly executed until it is determined in step S15 that the final drop position is reached. If the main control unit 50 determines that it is the final dropping position (step S15: YES), it ends the process.
• main control unit 50 performs steps S10 to S15 on all wells 12 included in the MEA plate 10 while changing the wells 12 to be seeded.
• the electrode position is detected based on the image obtained by imaging the bottom surface 14 of the well 12, and the dropping position is determined based on the detected information on the electrode position. , cells can be seeded with high precision.
• FIG. 8 shows an example of the bottom surface 14 of the well 12 onto which the droplet DL has been dropped by the cell seeding device 2.
• Each droplet DL contains one or more cells. After the droplet DL is dropped on the bottom surface 14, the droplet DL becomes flat due to its own weight, thereby increasing its planar shape (that is, expanding). Further, the cells contained in the droplet DL are adhesive cells and adhere to the measurement electrode 31.
• FIG 9 shows the evaluation flow for toxicity evaluation.
• the MEA plate 10 is set in the cell seeding device 2, and cells are seeded into each well 12 (step S20).
• the MEA plate 10 is taken out from the cell seeding device 2, and after a culture solution is injected into each well 12, culture is performed in the culture device (step S21). Cultivation is carried out for a period of about one week. By culturing, the seeded cells wake up, and a plurality of cells bond with each other to form a cell group exhibiting a single behavior (for example, a myocardial sheet that beats spontaneously).
• step S22 the MEA plate 10 is taken out from the culture device, and the extracellular potential is measured by the potential measuring device (step S22).
• step S22 a drug candidate compound is added to the culture solution, and toxicity evaluation is performed by analyzing changes in the waveform before and after the addition of the drug candidate compound.
• the dropping position is determined based on the detection information of the electrode position, but in the first modification, the dropping amount is determined in addition to the dropping position.
• FIG. 10 shows functions configured in the processor 40 and controller 5 according to the first modification.
• the processor 40 includes a main control section 50, an image acquisition section 51, an electrode position detection section 52, and a drop position determination section 53, as well as a drop amount determination section 54.
• the dropping amount determining unit 54 determines the dropping amount of the droplet DL (that is, the volume of the droplet DL) by the dropping unit 21 based on the detection information of the electrode position detected by the electrode position detecting unit 52.
• the dropping amount determination unit 54 calculates the shortest distance L between the measurement electrode 31 and the reference electrode 33 based on the detection information of the electrode position. Then, based on the shortest distance L, the dropped amount determining unit 54 determines the amount of the dropped droplet DL that will not expand and reach the reference electrode 33.
• a reference table 55 is stored in the storage unit 41.
• the reference table 55 stores the relationship between the droplet amount and the enlarged size of the droplet DL.
• the drop amount and the radius after expansion of the droplet DL (hereinafter referred to as expansion radius) are stored.
• the dropping amount determining unit 54 After calculating the shortest distance L, the dropping amount determining unit 54 refers to the reference table 55 and determines the dropping amount at which the expansion radius of the droplet DL is a constant ratio of the shortest distance L.
• the main control unit 50 controls the volume of the droplet DL dropped from the dropping unit 21 by the dropping amount determining unit 54 by controlling the piezoelectric element of the dropping unit 21 via the dropping control unit 5A. Use the determined dripping amount.
• the other configuration of the cell seeding device according to the first modification is the same as the configuration of the cell seeding device 2 according to the above embodiment.
• the dropping amount is determined in addition to the dropping position based on the detection information of the electrode position, so cells can be seeded with higher accuracy.
• the reference table 55 may also store information (for example, standard deviation) representing variations in the expansion radius.
• the dropping amount determination unit 54 determines the dropping amount by taking into account the variation in the expansion radius. For example, the dropping amount determining unit 54 determines the dropping amount such that the value obtained by adding the standard deviation to the expansion radius (average value) is a constant ratio of the shortest distance L.
• the expansion radius of the droplet DL changes depending on the contact angle between the droplet DL and the bottom surface 14 of the well 12.
• the contact angle refers to the angle between the tangent of the droplet DL and the bottom surface 14. The smaller the contact angle, the larger the expansion radius.
• the contact angle varies depending on the main components of the cell suspension, the type of coating on the bottom surface 14, etc.
• the reference table 55 may store a plurality of enlargement radii according to information related to the contact angle. Further, information related to the contact angle may be inputted via the input unit 42, and the main control unit 50 may correct the enlargement radius stored in the reference table 55 based on the input information.
• the expansion radius is not limited to the data stored in the reference table 55, and may be defined as a function that depends on the amount of dripping. Further, the radius of expansion may be defined as a function depending on information related to the drop amount and the contact angle.
• the drop amount is determined in addition to the drop position based on the detection information of the electrode position, but in the second modification, the determined drop amount can be further changed by the user.
• FIG. 13 shows functions configured in the processor 40 and controller 5 according to the second modification.
• the second modification differs from the first modification in that the main control section 50 includes a drop amount changing section 56.
• the dropping amount changing unit 56 acquires the dropping amount determined by the dropping amount determining unit 54 and displays the expected enlarged size of the droplet DL based on the determined dropping amount via the display unit 43. Present to the user. The user can change the dripping amount using the input unit 42. The dripping amount changing unit 56 changes the dripping amount to be dripped by the dripping portion 21 when an operation to change the dripping amount is performed.
• FIG. 14 shows an example of a GUI (Graphical User Interface) screen 60 displayed on the display unit 43.
• the dropping amount changing unit 56 causes the display unit 43 to display a GUI screen 60 shown in FIG. 14 in order to present the expected enlarged size of the droplet DL and to change the dropping amount.
• the GUI screen 60 includes an image display area 61, a change operation area 62, and a decision button 63.
• the dropping amount changing unit 56 displays the image acquired by the image acquiring unit 51 in the image display area 61. Further, the droplet amount changing unit 56 displays an area 70 (hereinafter referred to as an assumed area) of the droplet DL that is expected to be enlarged based on the droplet amount determined by the droplet amount determining unit 54. In this modification, the assumed area 70 is circular. The droplet amount changing unit 56 determines the size of the assumed area 70 based on the expansion radius of the droplet DL acquired by the droplet amount determining unit 54 from the reference table 55 described above.
• the dripping amount changing unit 56 causes the changing operation area 62 to display a plurality of dripping amounts including the dripping amount determined by the dropping amount determining unit 54. By operating the cursor 64, the user can perform a selection operation to select the drop amount. When a drop amount is selected, the drop amount changing unit 56 changes the size of the assumed area 70 to a size corresponding to the selected drop amount. The user can select a drop amount to make the expected area 70 a desired size while checking the size of the expected area 70. The user can perform a determination operation to determine the final dripping amount by clicking the determination button 63.
• FIG. 15 shows an example of the flow of the dropping amount changing process by the dropping amount changing unit 56.
• the dropping amount changing unit 56 acquires the dropping amount determined by the dropping amount determining unit 54 (step S20). Furthermore, when determining the droplet amount, the enlargement radius of the droplet DL obtained by the droplet amount determination unit 54 is obtained from the reference table 55 (step S21).
• the dropping amount changing unit 56 generates a GUI screen 60 shown in FIG. 14 based on the droplet amount obtained in step S20 and the enlargement radius of the droplet DL obtained in step S21, and displays it on the display unit 43. (Step S22).
• the dripping amount changing unit 56 determines whether the user has performed a selection operation to select the dripping amount based on the GUI screen 60 (step S23).
• step S23 If there is a selection operation (step S23: YES), the drop amount changing unit 56 changes the size of the assumed area 70 to a size corresponding to the selected drop amount (step S24). If there is no selection operation (step S23: NO), the dripping amount changing unit 56 determines whether or not there is a determination operation (step S25).
• step S25 NO
• step S25: YES the dropping amount changing unit 56 changes the dropping amount (step S26) and ends the process. Note that the droplet amount changing unit 56 does not change the droplet amount when the droplet amount selected by the user is the droplet amount determined by the droplet amount determining unit 54.
• the main control unit 50 controls the volume of the droplet DL dropped from the dropping unit 21 by controlling the piezoelectric element of the dropping unit 21 via the dropping control unit 5A, so that the volume of the droplet DL dropped from the dropping unit 21 is controlled by the dropping amount changing unit 56. The dripping amount is changed.
• the droplet amount is determined in addition to the droplet position based on the detection information of the electrode position, and the user can further change the droplet amount, so cells can be seeded with more precision. can.
• the variation is The assumed area of the considered size may be displayed on the display unit 43.
• an assumed area 70 corresponding to the enlarged radius (average value), an assumed area 71 corresponding to the radius obtained by adding the standard deviation ⁇ to the enlarged radius (average value), and an assumed area 71 corresponding to the enlarged radius (average value) ) may be displayed on the display unit 43 on the assumption area 72 corresponding to the radius obtained by subtracting the standard deviation ⁇ .
• the user may select which of the assumed areas 70, 71, and 72 to display.
• a selection operation area 65 is provided on the GUI screen 60 in which variations in the enlargement radius can be selected.
• the main control unit 50 selects the assumed area 70 when “0” is clicked, selects the assumed area 71 when “+ ⁇ ” is clicked, and selects the assumed area 72 when “ ⁇ ” is clicked. do.
• FIG. 17 illustrates a case where "+ ⁇ " is clicked. Thereby, the user can determine the drop amount while checking the degree of variation in the enlargement radius.
• the imaging device 4 is an image sensor that captures images using visible light. Instead, the imaging device 4 uses a laser beam that performs imaging by irradiating the bottom surface 14 of the well 12 with a laser beam and receiving the reflected laser beam reflected by the electrodes including the measurement electrode 31 and the reference electrode 33. It may be a sensing device based on the above-mentioned method.
• the dispenser 3 is of a piezoelectric type, but is not limited to the piezoelectric type, and a pressure type dispenser or the like may be used. Furthermore, the dropping device of the present disclosure includes a so-called bio-3D printer.
• a processing unit that executes various processes such as the main control unit 50, image acquisition unit 51, electrode position detection unit 52, dripping position determining unit 53, dripping amount determining unit 54, and dripping amount changing unit 56 described above.
• the hardware structure is, for example, various processors as shown below.
• processors include CPUs, programmable logic devices (PLDs), dedicated electric circuits, and the like.
• a CPU is a general-purpose processor that executes software (programs) and functions as various processing units.
• a PLD is a processor such as an FPGA (Field Programmable Gate Array) whose circuit configuration can be changed after manufacturing.
• the dedicated electric circuit is a processor, such as an ASIC (Application Specific Integrated Circuit), that has a circuit configuration specifically designed to execute a specific process.
• ASIC Application Specific Integrated Circuit
• One processing unit may be composed of one of these various processors, or may be composed of a combination of two or more processors of the same type or different types (for example, multiple FPGAs or a combination of a CPU and FPGA). may be done. Further, the plurality of processing units may be configured with one processor.
• First as an example of configuring a plurality of processing units with one processor, there is a configuration in which a single processor is configured by a combination of one or more CPUs and software, and this processor functions as a plurality of processing units.
• SoC system on chip
• circuitry that is a combination of circuit elements such as semiconductor elements.
• the present disclosure is not limited to the embodiments described above, and that various configurations can be adopted as long as they do not depart from the gist of the present disclosure. Further, the present disclosure extends to a computer-readable storage medium that non-temporarily stores the program in addition to the program.
• a dropping device that drops droplets of cell suspension onto a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom; an imaging device that is provided in the dropping device and that captures an image of the bottom surface to generate an image; The droplet detects the positions of the measurement electrode and the reference electrode based on the image, and makes it possible to place the cell on the measurement electrode without placing the cell on the reference electrode based on the detection information.
• an information processing device that determines the dropping position of the A cell seeding device comprising: [Additional note 2] The information processing device determines the dropping position and the dropping amount of the droplet based on the detection information.
• the cell seeding device according to Supplementary Note 1.
• the information processing device determines the dropping amount at which the dropped droplet will not expand and reach the reference electrode, based on the relationship between the dropping amount and the size of the droplet after expansion.
• the information processing device presents to the user an assumed enlarged size of the droplet based on the determined droplet amount, and changes the droplet amount based on an operation by the user.
• the imaging device is an image sensor that captures images using visible light, or a laser-based sensing device.
• the reference electrode is arranged around a microelectrode array in which a plurality of the measurement electrodes are arranged in a two-dimensional array.
• the cell seeding device according to any one of Supplementary Notes 1 to 5.
• the cells are cardiomyocytes or nerve cells, The cell seeding device according to any one of Supplementary Notes 1 to 6.
• An information processing device that determines the amount of droplets of a cell suspension to be dropped by a dropping device into a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom, Equipped with a processor, The processor detects the positions of the measurement electrode and the reference electrode based on an image generated by imaging the bottom surface with an imaging device, and the processor detects the positions of the measurement electrode and the reference electrode without placing cells on the reference electrode based on the detection information. determining a dropping position of the droplet that allows the cell to be placed on the measurement electrode; Information processing device.
• An information processing method for determining the amount of a droplet of a cell suspension to be dropped by a dropping device into a cell culture vessel in which a measurement electrode and a reference electrode are arranged on the bottom surface comprising: An imaging device detects the positions of the measurement electrode and the reference electrode based on an image generated by imaging the bottom surface, and based on the detection information, the measurement electrode is placed on the measurement electrode without placing a cell on the reference electrode. determining a dropping position of the droplet that allows cells to be deposited; Information processing method.

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