WO2023153414A1 - System, program and method for evaluating sheeted state of sheet-shaped cell culture, and method for producing sheet-shaped cell culture - Google Patents

System, program and method for evaluating sheeted state of sheet-shaped cell culture, and method for producing sheet-shaped cell culture Download PDF

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Publication number
WO2023153414A1
WO2023153414A1 PCT/JP2023/004074 JP2023004074W WO2023153414A1 WO 2023153414 A1 WO2023153414 A1 WO 2023153414A1 JP 2023004074 W JP2023004074 W JP 2023004074W WO 2023153414 A1 WO2023153414 A1 WO 2023153414A1
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sheet
cells
culture
cell
cell culture
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PCT/JP2023/004074
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French (fr)
Japanese (ja)
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沙紀 河野
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テルモ株式会社
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    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a system, program, method, and manufacturing method for evaluating the sheet state of a sheet-like cell culture.
  • Non-Patent Document 1 a cell structure formed using a scaffold and a sheet-like cell culture in which cells are formed into a sheet have been developed.
  • Patent Document 1 describes a system for determining whether a sheet-like cell culture has been formed correctly.
  • a system includes a storage unit for storing a culture vessel for sheet-forming culture of sheet-forming cells, a measurement unit for measuring the concentration of non-adherent cells in a liquid medium or a change in the concentration, and the measurement value measured by the measurement unit. It is described that formation of a sheet-like cell culture is determined by measuring the concentration of non-adherent cells in a liquid medium, including an analysis unit that analyzes and calculates the non-adherent cell ratio.
  • Patent Document 2 describes a system for determining whether a sheet-like cell culture has been formed correctly.
  • a system includes a storage unit for storing a culture vessel for sheet-forming culture of sheet-forming cells, a measurement unit for measuring the state of adhesion between the sheet-forming cells and the culture vessel, and analysis of the results obtained by the measurement unit. It is described that the sheet-forming state is determined by measuring the adhesion state between the sheet-forming cells and the culture vessel and including an analysis unit that calculates the adhesion rate.
  • an object of the present invention is to solve such problems and to provide a means for accurately evaluating the sheet-forming state of a sheet-like cell culture with a simple mechanism and simple operations.
  • the inventors of the present invention have found for the first time that it is possible to accurately evaluate the sheet-forming state of a sheet-like cell culture by adopting a simple mechanism in the course of intensive research to solve the above problems. As a result of further research based on the results, the present invention was completed.
  • a system for evaluating the sheet state of a sheet-shaped cell culture comprising: a measurement unit for acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded;
  • the measurement unit includes an analysis unit that analyzes the information obtained and calculates the degree of detachment of the sheet-shaped cell culture, and the measurement unit measures the inside of the culture container from the bottom direction and / or side direction that is in contact with the cell suspension of the culture container.
  • the system wherein the system is configured to measure cells of [2]
  • [3] The system according to [1] or [2], wherein the measurement is performed by light detection.
  • the measurement unit is a light projector and a light receiver, the light projector is arranged on the bottom or side surface of the culture vessel, the light receiver is arranged on the side or bottom surface of the culture vessel, and the measurement is performed from the light projector to the culture of the sheet-like cell culture.
  • the analysis unit is configured to determine that detachment processing of the sheet-like cell culture is necessary when the calculated value is equal to or greater than the set value System as described.
  • a program for evaluating the sheet state of a sheet-like cell culture comprising a process of acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded, and The processing to analyze the information and calculate the degree of detachment of the sheet-shaped cell culture is executed by a processor, and the processing to obtain is performed by analyzing the information from the bottom direction and / or side direction in contact with the cell suspension of the culture vessel.
  • the above program including a process of measuring cells in the container.
  • a method for evaluating the sheet state of a sheet-like cell culture comprising: obtaining information about the distribution of cells in a culture vessel in which a cell suspension is seeded; Including the step of analyzing the information to calculate the degree of detachment of the sheet-like cell culture, and the obtaining step includes: The above method, comprising measuring the cells.
  • a method for producing a sheet-like cell culture comprising: acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded; calculating the degree of detachment of the culture; and determining that detachment of the sheet-like cell culture is necessary when the calculated value is equal to or greater than a set value.
  • the manufacturing method comprising the step of measuring cells in the culture vessel from the bottom direction and/or the side direction in contact with.
  • the present invention it is possible to reliably acquire information on cell migration and shape change, so it is possible to evaluate the sheet state of sheet-like cell cultures with higher accuracy than before.
  • the present invention since analysis that is absolutely impossible by human observation or the like can be performed, it becomes possible to quantify the state of sheeting, which has been impossible in the past.
  • FIG. 1A shows a conceptual diagram of one aspect of the system of the present invention.
  • FIG. 1B shows a conceptual diagram of one aspect of the system of the present invention.
  • FIG. 2 is an explanatory diagram showing how the detachment of the peripheral portion of the sheet-shaped cell culture is detected.
  • FIG. 3 is a schematic diagram showing a configuration example of the system of the present invention.
  • FIG. 4 is a block diagram showing a configuration example of a terminal.
  • FIG. 5 shows a flow diagram of one aspect of the system of the present invention.
  • FIG. 6 shows a flow diagram of one aspect of the system of the present invention.
  • sheet-like cell culture refers to a sheet-like cell formed by connecting cells to each other.
  • Cells may be connected to each other directly (including through cellular elements such as adhesion molecules) and/or through intermediaries.
  • the intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include extracellular matrix.
  • the mediator is preferably of cell origin, in particular of cells that make up the cell culture.
  • the cells are at least physically (mechanically) linked, but may also be functionally linked, eg chemically, electrically.
  • the sheet-like cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminated (multilayered) body, e.g., two layers, three layers, 4 layers, 5 layers, 6 layers, etc.).
  • the sheet-like cell culture may have a three-dimensional structure in which the cells do not exhibit a clear layered structure and have a thickness exceeding the thickness of a single cell.
  • the cells may be non-uniformly arranged (for example, in a mosaic pattern) without being evenly aligned in the horizontal direction.
  • the sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds are sometimes used in the art to attach cells onto and/or into their surfaces and maintain the physical integrity of sheet-like cell cultures.
  • the sheet-like cell culture of the present invention can maintain its physical integrity without such a scaffold.
  • the sheet-like cell culture of the present invention can be coated with fibrin or the like to reinforce its physical strength.
  • adherent cells include adherent somatic cells and the like.
  • somatic cells include myoblasts (e.g., skeletal myoblasts), muscle satellite cells, mesenchymal stem cells (e.g., bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, intrauterine tissue stem cells such as cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, synovial cells, chondrocytes , Epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g.,
  • Somatic cells may be those differentiated from iPS cells (iPS cell-derived cells), iPS cell-derived cardiomyocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, and the like.
  • cell suspension refers to a liquid medium in which cells are suspended.
  • a liquid medium any medium commonly used for manipulation of cells, including but not limited to, balanced salt solutions such as saline, Ringer's solution, Hank's balanced salt solution, PBS (phosphate buffered saline), liquid A medium or the like can be used.
  • a sheet-like cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, JP-A-2010-081829, JP-A-2011-110368, etc.).
  • a method for producing a sheet-like cell culture typically includes the step of seeding cells (cell suspension) in a culture vessel at a density that allows formation of a sheet-like cell culture without substantial proliferation, seeding It includes, but is not limited to, forming a sheet from the cells that have been cultured, and peeling off the formed sheet-like cell culture from the culture vessel. Each of these steps can be performed by any known method suitable for producing a sheet-like cell culture.
  • the present invention may comprise a step of producing a sheet-like cell culture, wherein the step of producing a sheet-like cell culture comprises producing a sheet-like cell culture using the above system. good.
  • seeding a cell suspension refers to seeding a cell suspension in a culture vessel
  • sheet culture refers to a cell suspension seeded in a culture vessel (cell ) to form a sheet-like cell culture.
  • cells in a cell suspension seeded in a culture vessel float in a liquid medium immediately after seeding, and gradually settle to the bottom of the culture vessel over time. . Then, the cells adhere to the bottom surface of the culture vessel to form a part of the sheet-like cell culture, and the cells adhere to each other to form a sheet, thereby forming a sheet-like cell culture.
  • cells floating in a liquid medium are sometimes referred to as “non-adherent cells”
  • cells adhering to the bottom surface of a culture vessel are sometimes referred to as "adherent cells”.
  • cell distribution refers to the distribution of cells in a cell suspension.
  • Cells in a cell suspension are spherical immediately after being seeded and float in a liquid medium, and are distributed in a state in which the cells are separated from each other (the cells do not adhere to each other). It can be used as an index for evaluating the unformed state of the culture.
  • the distribution of cells on the part above the bottom surface of the culture vessel decreases, while the distribution of cells on the bottom surface increases. The change can be used as an index for evaluating the formation start state of the sheet-like cell culture.
  • the cells in the detached part are floating, and the detached cells have the property of gradually returning to their original shape (spherical), so they shrink further. These can be used as indicators for evaluating the detachment state of sheet-like cell cultures.
  • the analysis unit of the present invention can calculate the density distribution of cells in the cell suspension from the distribution of cells in the cell suspension.
  • the analysis unit three-dimensionally captures the cell distribution (cell movement) in the cell suspension and calculates the density distribution of the cells to analyze the progress of sheet-like cell culture. be able to. That is, since the floating, sedimentation, adhesion, and detachment of cells in a cell suspension correspond to the progress of sheet-like cell culture in sheet-like cell culture, the distribution of cells at each site in the cell suspension can be used as an index for sheet culture.
  • Each part in the cell suspension is represented by an x, y, z coordinate system when the cell suspension is three-dimensionally captured, such as the bottom of the culture vessel, above the bottom, the center of the bottom, and the periphery of the bottom. be able to.
  • the produced sheet-like cell culture will be uneven.
  • the unevenness referred to here means that the sheet-shaped cell culture has portions with different thicknesses (non-uniform thickness portions).
  • the sheet-like cell culture may be torn or damaged due to non-uniform thickness (unevenness) before use. Therefore, in producing a sheet-like cell culture, it is necessary to measure the uniformity of cell distribution on the bottom surface of the culture vessel to avoid such unevenness.
  • the “sheet state” quantitatively indicates the state of the sheet-like cell culture, including the degree of formation of the sheet-like cell culture and/or the degree of detachment of the sheet-like cell culture. , can be uniquely determined from the distribution of the cells in the cell suspension.
  • the "formation degree of the sheet-like cell culture” is not limited to this, but may be, for example, a degree of completion expressed as a percentage when the completed state of the sheet-like cell culture is 100%. , the degree of perfection equal to or higher than the set value may be set as "accepted”. That is, the degree of sheet-like cell culture formation can be quantified by calculating from the distribution of cells associated with floating, sedimentation, and adhesion of cells in the measured cell suspension.
  • the "degree of detachment of the sheet-like cell culture” can be quantified, for example, by measuring the area ratio of the part of the sheet-like cell culture adhered to the bottom of the culture vessel and the detached part.
  • the degree of detachment of the sheet-like cell culture is, for example, 0% (no detachment) when the entire sheet-like cell culture in a completed state of 100% is adhered to the bottom surface of the culture vessel, and a part is A state in which 1% of the area is in the liquid medium due to natural peeling is defined as 1% (1% peeling), which can be quantified.
  • the degree of detachment can be calculated by calculating the area of the naturally detached portion with respect to the bottom surface.
  • the sheet-shaped cell culture naturally detaches, the cells shrink and the detached part becomes larger. If left as it is, the sheet-shaped cell culture may curl up and become unsuitable for shipment.
  • the spontaneous detachment of the cells progresses while the sheet-like cell culture is incomplete, the sheet-like cell culture may not be formed well and may be unsuitable for shipment. Therefore, when the degree of detachment of the sheet-like cell culture reaches or exceeds a set value, detachment treatment (forced detachment) is preferably performed.
  • a set value can be freely set, for example, when the degree of peeling is 5%.
  • the sheet state numerical changes can be calculated by measuring a series of cell distribution states such as floating, sedimentation, adhesion, and detachment of cells in a cell suspension over time. For example, by setting the completed state of the sheet-shaped cell culture to 100% and the degree of peeling to 1%, it can be defined that the sheet-formed state has progressed 101%. Then, for example, when the sheet-formed state exceeds a first set value (e.g., 100% or more), it is determined that the sheet-like cell culture is completed, and the second set value (e.g., 101% or more) ), it can be determined that the sheet-like cell culture needs to be peeled off. In addition, detachment of cells during sheet culture (of a sheet-like cell culture before completion) can also be determined as abnormal detachment.
  • a first set value e.g., 100% or more
  • the second set value e.g. 101% or more
  • the sheet state before the sheet state exceeds a first set value (e.g., 100% or more) (e.g., when it is 90%), if the sheet-like cell culture begins to spontaneously detach, the sheet state It can also be determined as a defective product in which peeling occurred at 90%. Furthermore, by analyzing numerical changes in the sheet-formed state (for example, changes in the degree of completion), it is possible to predict the timing at which the sheet-shaped cell culture is completed and the timing at which spontaneous detachment begins. These predictions may be made by an operator, but the precision can be improved by comparing with a calibration curve prepared in advance. Accuracy can be further improved by using a trained model generated by machine learning or the like.
  • a first set value e.g., 100% or more
  • the "measurement part” refers to a part that acquires information on the distribution of cells in the culture vessel in which the cell suspension is seeded.
  • the measurement unit typically includes a photodetector, and the photodetector is not limited and includes, for example, a photoelectric sensor, a fiber sensor, a laser sensor, a color sensor, and the like.
  • a device can be included that can measure the distribution with photodetection.
  • the measurement unit of the present invention measures cells in the culture vessel from the bottom direction and / or side direction in contact with the cell suspension of the culture vessel. can be configured as
  • "measure cells from the portion of the culture vessel that is in contact with the cell suspension” means that the cells present in the cell suspension in the culture vessel are measured from the liquid surface of the cell suspension. , refers to measuring the cells in the culture vessel from the bottom direction and/or the side direction in contact with the cell suspension of the culture vessel. Since the moisture as described above is generated from the liquid surface of the cell suspension, the measuring unit in the present invention measures the humidity inside the culture vessel from the bottom direction and/or the side direction, which is in contact with the cell suspension of the culture vessel. By measuring the cells of the liquid, it is possible to avoid the influence of moisture generated from the liquid surface.
  • Cells in the culture vessel can also be measured by attaching the measurement section to the side or bottom of the culture vessel (without separating the measurement section from the culture vessel).
  • a photodetector for example, a method of measuring the light reflected from the cell with a light emitter and receiver (reflection type), or placing the light emitter and light receiver so that the cell is sandwiched between them allows the cell to pass through.
  • Cells can be measured by various methods and arrangements, such as a method of measuring transmitted light (transmissive type), and a method of measuring reflected light by placing a cell between a light emitter and a reflector (retroreflective type). can be measured.
  • the measurement side of the measurement unit can be placed on the bottom and/or side of the culture vessel.
  • the non-measurement side of the measurement unit can be placed anywhere, but is preferably placed on the side and/or bottom side of the culture vessel, which is less susceptible to moisture.
  • the system of the present invention adopts a simple mechanism in which the measurement unit is arranged in the direction of the bottom surface and/or the side surface in contact with the cell suspension of the culture vessel, thereby enabling sheet-like cell culture. It is possible to accurately evaluate the sheeting state of an object. For example, by measuring cells from the side of the culture vessel, it is possible to measure the movement (floating, sedimentation, detachment, etc.) and distribution of cells in the vertical direction (z-axis direction) without being affected by humidity. In addition, by measuring cells from the bottom side of the culture vessel, it is possible to measure the movement (adhesion, detachment, etc.) and distribution of cells in the horizontal direction (x-axis direction, y-axis direction) without being affected by humidity.
  • the measurement unit performs measurement so as to three-dimensionally cross the culture vessel diagonally, such as from the side of the culture vessel to the bottom direction and/or from the bottom side to the side direction, so that the vertical direction (z Axial) and horizontal (x-axis, y-axis) movements can also be measured simultaneously.
  • the movement of cells in the cell suspension in the vertical direction and/or the horizontal direction can be measured three-dimensionally for each region. It is advantageous for measuring the distribution of cells because it can be measured in a specific manner. That is, in the conventional system, cells are measured as two-dimensional information per area of the bottom surface of the culture vessel, whereas in the system of the present invention, cells can be measured as three-dimensional information per volume of the cell suspension. , the sheeting state can be evaluated more accurately.
  • a photodetector is used as the measurement unit, information on the distribution of cells in the cell suspension can be measured as information on the transmittance and reflectance of light, so advanced techniques such as image analysis are not required. analysis cost can be kept low.
  • the "analysis part” refers to a part that analyzes the information acquired by the measurement part and calculates the degree of formation and/or the degree of detachment of the sheet-like cell culture.
  • the analysis unit can include a processor or the like for analyzing information obtained by the measurement unit.
  • the information acquired by the measurement unit includes information on cell distribution, and such information can include, for example, information on light transmittance and reflectance for cells at each site in the cell suspension. In other words, the light transmittance and reflectance are different between areas where a large number of cells are distributed and areas where a small number of cells are distributed in the cell suspension, and the cell density distribution is calculated by analyzing these information. It is possible to evaluate the sheeting state. It is also possible to analyze the information from the measurement unit over time, and graph the changes in the sheet-formed state of the sheet-like cell culture based on the change over time.
  • the analysis unit can quantify the sheet-forming state (degree of formation and/or degree of detachment) of the sheet-shaped cell culture and output such calculated values in real time. Further, when the calculated value is the first set value or more, it is determined that the sheet-like cell culture is completed, and when the calculated value is the second set value or more, the sheet-like cell culture is naturally exfoliated. It can also be determined that That is, the analysis unit can output, for example, information regarding calculation and determination in real time. Information related to calculation and determination can also include predictions (time, countdown, etc.) regarding the timing of completion, the timing of natural peeling, and the timing of peeling processing (forced peeling).
  • Formation and/or peeling may be determined by the operator based on the numerical value of the sheeting state, or by the system comparing the numerical value with the set value.
  • the determination by the analysis unit can also be performed using a calibration curve prepared in advance or a learned model.
  • a trained model is created by removing missing values and outliers and extracting correct data from raw data in a sheet state derived from cell counting and image analysis by an expert, for example. , generate a training data set, and generate learned parameters as a result of learning using the training data set.
  • the correct answer data can include knowledge based on experiences of experts regarding cell uniformity, thickness unevenness, sheet state, peeling, and the like.
  • a trained model is generated as an inference program that incorporates the learned parameters, i.e., a program that can output a certain result for the input by applying the built-in learned parameters. can do.
  • a trained model that can be suitably used for the information measured by the photodetector is generated. can do.
  • various patterns of sheet culture can be recognized by analyzing the information measured by the photodetector.
  • a person skilled in the art can combine various learning data, learning methods, and learning models to generate an optimal learning model.
  • the system of the present invention includes at least a measurement unit, but may further include an analysis unit, a storage unit, an input unit, an output unit, and the like.
  • the storage unit is a part that stores information acquired by the measurement unit, information analyzed by the analysis unit, programs for operating the system, etc.
  • Various electronic storage media such as semiconductor memory, hard disk, cloud storage etc.
  • the input part is a part where the operator of the system or an external system inputs measurement parameters and programs, and various input interfaces, for example, means for receiving signals such as electricity and light from other systems , connectors, wireless communication devices, etc.), buttons, keyboards, touch panels, and the like.
  • the output section is a section that outputs predetermined signals and information based on the information acquired by the measurement section and the information analyzed by the analysis section, and outputs various output interfaces, such as electricity, light, and information.
  • means electric wire, optical fiber, connector, wireless communication device, etc.
  • display touch panel, printer, patrol lamp, buzzer, voice synthesizer, etc.
  • An input unit and an output unit may be integrated to be used as a general-purpose computer or a smart terminal (smartphone, tablet terminal).
  • the system of the present invention may include software for general-purpose computers and applications for smart terminals, and the information acquired by the measurement unit, the information analyzed by the analysis unit, etc. are used by email or application push notification. It can also be configured to send to
  • the system of the present invention may further include a peeling section for peeling the sheet-like cell culture. That is, as described above, detachment of the sheet-like cell culture (including completed and unfinished) results in a state unsuitable for shipment.
  • the peeling unit may be configured to perform peeling processing (forced processing).
  • a separation unit for example, means for vibrating liquid (see Japanese Patent Laid-Open No. 2016-52269) can be used.
  • the peeling section can further include a means for spreading the peeled sheet-like cell culture (see Japanese Patent Application Laid-Open No. 2016-52270).
  • the peeling portion is not limited to these, and various known means can be used or combined.
  • the system of the present invention includes at least a measurement section, and may further include an analysis section, a storage section, an input section, an output section, and the like.
  • FIG. 1 shows a conceptual diagram of one embodiment of the system of the present invention.
  • the system of the present invention includes a measurement unit 1, and the measurement unit 1 is a portion (side surface) in contact with the cell suspension L of the culture vessel C seeded with the cell suspension L. ), vertical (z-axis) and/or horizontal (x-axis, y-axis) migration of cells can be measured.
  • one measurement unit 1 can be arranged on the bottom surface of the culture container C, and six measurement units can be arranged so as to surround the side surface of the culture container C. As shown in FIG. This makes it possible to more accurately measure the cell distribution at each site in the cell suspension. By measuring the movement and distribution of cells in the vertical direction (z-axis direction), it is possible to analyze states and changes such as cell suspension, sedimentation, and detachment.
  • FIG. 2 is an explanatory diagram showing how the detachment of the peripheral portion of the sheet-shaped cell culture is detected.
  • the measurement unit 1 is a light projector (light source) 1a or a light receiver 1b.
  • the light projector 1a is arranged on the bottom surface of the culture container, and the light receiver 1b is arranged on the side surface of the culture container. Note that the correspondence relationship between the light projector 1a and the light receiver 1b may be reversed.
  • the peeling of the sheet-like cell culture S has not started, the light emitted obliquely from the light projector 1a to the culture surface of the sheet-like cell culture S is emitted from the sheet-like cell culture.
  • the vertical direction (z-axis direction) of the cells and the Movement in the horizontal direction (x-axis direction, y-axis direction) can also be measured at the same time.
  • Detachment of the peripheral edge of the sheet-like cell culture can be preferably measured by sandwiching the peripheral edge of the bottom surface of the culture vessel between the measuring instrument on the side surface and the measuring instrument on the bottom side.
  • FIG. 3 is a schematic diagram showing a configuration example of the system 100 of the present invention.
  • a system 100 includes a measurement unit 1 and a terminal 2 .
  • the terminal 2 is an information processing device capable of various information processing, such as a tablet terminal.
  • the tablet terminal functions as the analysis unit, the storage unit, the input unit, and the output unit described above by executing the programs installed in the tablet terminal.
  • FIG. 4 is a block diagram showing a configuration example of the terminal 2.
  • the terminal 2 includes a control section 21 , a main storage section 22 , a communication section 23 , a display section 24 , an input section 25 and an auxiliary storage section 26 .
  • the control unit 21 has an arithmetic processing unit such as one or more CPU (Central Processing Unit), MPU (Micro-Processing Unit), GPU (Graphics Processing Unit), etc., and executes the program P stored in the auxiliary storage unit 26. Various information processing, control processing, etc. are performed by reading and executing the data.
  • CPU Central Processing Unit
  • MPU Micro-Processing Unit
  • GPU Graphics Processing Unit
  • the main storage unit 22 is a temporary storage area such as SRAM (Static Random Access Memory) or DRAM (Dynamic Random Access Memory), and temporarily stores data necessary for the control unit 21 to perform arithmetic processing.
  • the communication unit 23 is a communication module for performing processing related to communication, and transmits and receives information to and from the outside.
  • the display unit 24 is a display screen such as a liquid crystal display, and displays images.
  • the input unit 25 is an operation interface such as a keyboard and a mouse, and receives operation inputs from the operator.
  • the auxiliary storage unit 26 is a nonvolatile storage area such as a hard disk, and stores programs P (program products) necessary for the control unit 21 to execute processing and other data.
  • the terminal 2 may be provided with a reading unit for reading a portable storage medium 2a such as a CD-ROM, and read and execute the program P from the portable storage medium 2a.
  • a reading unit for reading a portable storage medium 2a such as a CD-ROM, and read and execute the program P from the portable storage medium 2a.
  • FIG. 5 shows a flow diagram in one aspect of the system of the present invention.
  • the measurement unit measures the distribution of cells
  • the analysis unit calculates the degree of sheet-like cell culture formation
  • (F3) Output the calculated value to the output unit.
  • (F4) compare the calculated value and the set value, and (F5) if the calculated value does not exceed the set value, determine that the sheet-like cell culture is incomplete (no) and proceed to (F1) return.
  • the operator Based on the calculated value displayed on the output unit in (F3), the operator compares the calculated value with the set value (equivalent to the process of F4), determines whether the sheet-like cell culture is complete or incomplete (F5 (equivalent to the processing of ) may be performed manually.
  • the peeling process may be performed in the peeling section as described above, or may be manually performed by an operator.
  • the calculated value in (F3) may be the time or countdown of the completion of the sheet-like cell culture predicted using a calibration curve or a learning model, in which case the operator is required in advance after completion Be prepared to work.
  • system in the present invention can operate according to the flow diagram shown in FIG.
  • FIG. 6 shows a flow diagram in one aspect of the system of the present invention.
  • the measurement unit measures the distribution of cells
  • the analysis unit calculates the degree of detachment of the sheet-like cell culture
  • P3 Output the calculated value to the output unit
  • P4 compare the calculated value with the set value
  • P5 if the calculated value does not exceed the set value, detachment of the sheet-like cell culture is performed. It determines that it is unnecessary (no) and returns to (P1).
  • the set value of (P4) can be freely set as described above, such as when the completed state is 100%, when the peeling degree is 1%, and when the sheet state is 101%. Further, in (P2) and (P3), in addition to the degree of peeling, the degree of formation can also be calculated and output. In (P6), abnormal peeling or incompleteness (completeness 90%) may be output as information.
  • the operator Based on the calculated value output to the output unit in (P3), the operator compares the calculated value with the set value (equivalent to the process of P4), determines the detachment process of the sheet-like cell culture (P5 (equivalent to the processing of ) may be performed.
  • the peeling process may be performed in the peeling section as described above, or may be manually performed by an operator.
  • the calculated value in (P3) may be the time to start the detachment treatment of the sheet-like cell culture predicted from the measurement over time, in which case the operator prepares the work necessary for the detachment treatment in advance. It can be performed.
  • the sheet-like cell culture begins to gradually peel off from the peripheral edge, so the measurement by the measurement unit should be performed at least on the peripheral edge of the bottom surface of the culture vessel. That is, as described above, the measurement unit performs measurement so as to obliquely cross the periphery of the bottom surface of the culture vessel, such as from the side surface of the culture vessel toward the bottom surface and/or from the bottom surface side toward the side surface. , the detachment of the sheet-like cell culture can also be detected as a movement that physically disappears from the field of view.

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Abstract

Provided is a system for evaluating the sheeted state of a sheet-shaped cell culture, said system including: a measurement unit for acquiring information about the distribution of cells in a culture container in which a cell suspension is seeded; and an analysis unit for analyzing the information acquired by the measurement unit and calculating the degree of detachment of the sheet-shaped cell culture. The measurement unit is configured to measure the cells in the culture container from the bottom and/or side directions of the culture container in contact with the cell suspension.

Description

シート状細胞培養物のシート化状態を評価するためのシステム、プログラム、方法及びシート状細胞培養物の製造方法System, program, method for evaluating sheet-like state of sheet-like cell culture, and method for producing sheet-like cell culture
　本発明は、シート状細胞培養物のシート化状態を評価するためのシステム、プログラム、方法及び製造方法に関する。 The present invention relates to a system, program, method, and manufacturing method for evaluating the sheet state of a sheet-like cell culture.
　近年、損傷した組織等の修復のために、種々の細胞を移植する試みが行われている。例えば、狭心症、心筋梗塞などの虚血性心疾患により損傷した心筋組織の修復のために、胎児心筋細胞、骨格筋芽細胞、間葉系幹細胞、心臓幹細胞、ＥＳ細胞、ｉＰＳ細胞等の利用が試みられている（非特許文献１）。
　このような試みの一環として、スキャフォールドを利用して形成した細胞構造物や、細胞をシート状に形成したシート状細胞培養物が開発されてきた（非特許文献２）。 In recent years, attempts have been made to transplant various cells in order to repair damaged tissues and the like. For example, use of fetal myocardial cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for the repair of myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. has been attempted (Non-Patent Document 1).
As part of such attempts, a cell structure formed using a scaffold and a sheet-like cell culture in which cells are formed into a sheet have been developed (Non-Patent Document 2).
　特許文献１には、シート状細胞培養物が正しく形成されたか否かを判定するためのシステムが記載されている。かかるシステムは、シート形成細胞をシート化培養する培養容器を収納する収納部、液体培地中の非接着細胞濃度またはその変化を測定するための測定部、および前記測定部で測定された測定値を解析して非接着細胞率を算出する解析部を含み、液体培地中の非接着細胞濃度を測定することで、シート状細胞培養物の形成を判定することが記載されている。 Patent Document 1 describes a system for determining whether a sheet-like cell culture has been formed correctly. Such a system includes a storage unit for storing a culture vessel for sheet-forming culture of sheet-forming cells, a measurement unit for measuring the concentration of non-adherent cells in a liquid medium or a change in the concentration, and the measurement value measured by the measurement unit. It is described that formation of a sheet-like cell culture is determined by measuring the concentration of non-adherent cells in a liquid medium, including an analysis unit that analyzes and calculates the non-adherent cell ratio.
　特許文献２には、シート状細胞培養物が正しく形成されたか否かを判定するためのシステムが記載されている。かかるシステムは、シート形成細胞をシート化培養する培養容器を収納する収納部、シート形成細胞と培養容器との接着状態を測定するための測定部、および前記測定部によって得られた結果を解析して接着率を算出する解析部を含み、シート形成細胞と培養容器との接着状態を測定することで、シート化状態を判定することが記載されている。 Patent Document 2 describes a system for determining whether a sheet-like cell culture has been formed correctly. Such a system includes a storage unit for storing a culture vessel for sheet-forming culture of sheet-forming cells, a measurement unit for measuring the state of adhesion between the sheet-forming cells and the culture vessel, and analysis of the results obtained by the measurement unit. It is described that the sheet-forming state is determined by measuring the adhesion state between the sheet-forming cells and the culture vessel and including an analysis unit that calculates the adhesion rate.
特開２０１２－１５２１８８JP 2012-152188 特開２０１２－１５２１８９JP 2012-152189
　これまでのシステムは、液中の細胞の状態を観察することで、シート状細胞培養物のシート化状態を判定していたが、かかる観察には高度な技術が必要とされ、また、環境によっては観察が困難になるなどの問題が生じていた。したがって、本発明の目的は、このような問題を解決し、簡便な機構と、簡単な作業で、シート状細胞培養物のシート化状態を正確に評価するための手段を提供することにある。 Conventional systems have determined the sheet state of sheet-like cell cultures by observing the state of the cells in the liquid, but such observation requires advanced technology, and depending on the environment, had problems such as difficulty in observation. Accordingly, an object of the present invention is to solve such problems and to provide a means for accurately evaluating the sheet-forming state of a sheet-like cell culture with a simple mechanism and simple operations.
　本発明者らは、上記課題を解決するために鋭意研究を進める中で、簡単な機構を採用することで、シート状細胞培養物のシート化状態を正確に評価できることを初めて見出し、かかる知見に基づいてさらに研究を続けた結果、本発明を完成させるに至った。 The inventors of the present invention have found for the first time that it is possible to accurately evaluate the sheet-forming state of a sheet-like cell culture by adopting a simple mechanism in the course of intensive research to solve the above problems. As a result of further research based on the results, the present invention was completed.
　すなわち本発明は、以下に関する。
［１］シート状細胞培養物のシート化状態を評価するためのシステムであって、細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得する測定部、および測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出する解析部を含み、測定部は、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するように構成されている、前記システム。
［２］測定が、培養容器の側面方向から行われる、［１］に記載のシステム。
［３］測定が、光検出により行われる、［１］または［２］に記載のシステム。
［４］測定部は投光器及び受光器であり、投光器は培養容器の底面又は側面に配置され、受光器は培養容器の側面又は底面に配置され、測定が、投光器からシート状細胞培養物の培養面に対して斜めに照射された光を受光器で検出することにより行われる、［３］に記載のシステム。
［５］解析部が、算出値が設定値以上である場合にシート状細胞培養物の剥離処理が必要と判定するように構成されている、［１］～［４］のいずれか一つに記載のシステム。
［６］解析部による解析が、学習済みモデルを用いて行われる、［１］～［５］のいずれか一つに記載のシステム。
［７］解析部による解析結果を出力する出力部をさらに含む、［１］～［６］のいずれか一つに記載のシステム。 That is, the present invention relates to the following.
[1] A system for evaluating the sheet state of a sheet-shaped cell culture, comprising: a measurement unit for acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded; The measurement unit includes an analysis unit that analyzes the information obtained and calculates the degree of detachment of the sheet-shaped cell culture, and the measurement unit measures the inside of the culture container from the bottom direction and / or side direction that is in contact with the cell suspension of the culture container. The system, wherein the system is configured to measure cells of
[2] The system according to [1], wherein the measurement is performed from the lateral direction of the culture vessel.
[3] The system according to [1] or [2], wherein the measurement is performed by light detection.
[4] The measurement unit is a light projector and a light receiver, the light projector is arranged on the bottom or side surface of the culture vessel, the light receiver is arranged on the side or bottom surface of the culture vessel, and the measurement is performed from the light projector to the culture of the sheet-like cell culture. The system according to [3], which is carried out by detecting light emitted obliquely to the surface with a light receiver.
[5] Any one of [1] to [4], wherein the analysis unit is configured to determine that detachment processing of the sheet-like cell culture is necessary when the calculated value is equal to or greater than the set value System as described.
[6] The system according to any one of [1] to [5], wherein the analysis by the analysis unit is performed using a trained model.
[7] The system according to any one of [1] to [6], further including an output unit that outputs analysis results by the analysis unit.
［８］シート状細胞培養物のシート化状態を評価するためのプログラムであって、細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得する処理、および測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出する処理をプロセッサに実行させ、取得する処理は、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定する処理を含む、前記プログラム。 [8] A program for evaluating the sheet state of a sheet-like cell culture, comprising a process of acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded, and The processing to analyze the information and calculate the degree of detachment of the sheet-shaped cell culture is executed by a processor, and the processing to obtain is performed by analyzing the information from the bottom direction and / or side direction in contact with the cell suspension of the culture vessel. The above program, including a process of measuring cells in the container.
［９］シート状細胞培養物のシート化状態を評価するための方法であって、細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得するステップ、および測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出するステップを含み、取得するステップは、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するステップを含む、前記方法。 [9] A method for evaluating the sheet state of a sheet-like cell culture, comprising: obtaining information about the distribution of cells in a culture vessel in which a cell suspension is seeded; Including the step of analyzing the information to calculate the degree of detachment of the sheet-like cell culture, and the obtaining step includes: The above method, comprising measuring the cells.
［１０］シート状細胞培養物の製造方法であって、細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得するステップ、測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出するステップ、および算出値が設定値以上である場合にシート状細胞培養物の剥離処理が必要と判定するステップを含み、取得するステップは、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するステップを含む、前記製造方法。 [10] A method for producing a sheet-like cell culture, comprising: acquiring information about the distribution of cells in a culture vessel in which a cell suspension is seeded; calculating the degree of detachment of the culture; and determining that detachment of the sheet-like cell culture is necessary when the calculated value is equal to or greater than a set value. The manufacturing method, comprising the step of measuring cells in the culture vessel from the bottom direction and/or the side direction in contact with.
　本発明によれば、細胞の移動や形状変化に関する情報を確実に取得できるため、従来よりも高い精度でシート状細胞培養物のシート化状態を評価することができる。また、本発明によれば、人間による観察等によってでは到底不可能な解析ができるため、従来不可能であったシート化状態の数値化が可能となる。 According to the present invention, it is possible to reliably acquire information on cell migration and shape change, so it is possible to evaluate the sheet state of sheet-like cell cultures with higher accuracy than before. In addition, according to the present invention, since analysis that is absolutely impossible by human observation or the like can be performed, it becomes possible to quantify the state of sheeting, which has been impossible in the past.
図１Ａは、本発明のシステムの一態様における概念図を示す。FIG. 1A shows a conceptual diagram of one aspect of the system of the present invention. 図１Ｂは、本発明のシステムの一態様における概念図を示す。FIG. 1B shows a conceptual diagram of one aspect of the system of the present invention. 図２は、シート状細胞培養物の周縁部の剥離を検出する様子を示す説明図である。FIG. 2 is an explanatory diagram showing how the detachment of the peripheral portion of the sheet-shaped cell culture is detected. 図３は、本発明のシステムの構成例を示す模式図である。FIG. 3 is a schematic diagram showing a configuration example of the system of the present invention. 図４は、端末の構成例を示すブロック図である。FIG. 4 is a block diagram showing a configuration example of a terminal. 図５は、本発明のシステムの一態様におけるフロー図を示す。FIG. 5 shows a flow diagram of one aspect of the system of the present invention. 図６は、本発明のシステムの一態様におけるフロー図を示す。FIG. 6 shows a flow diagram of one aspect of the system of the present invention.
　本発明において「シート状細胞培養物」とは、細胞が互いに連結してシート状になったものをいう。細胞同士は、直接（接着分子などの細胞要素を介するものを含む）および／または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも物理的（機械的）に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、細胞培養物を構成する細胞に由来するものである。細胞は少なくとも物理的（機械的）に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。シート状細胞培養物は、１の細胞層から構成されるもの（単層）であっても、２以上の細胞層から構成されるもの（積層（多層）体、例えば、２層、３層、４層、５層、６層など）であってもよい。また、シート状細胞培養物は、細胞が明確な層構造を示すことなく、細胞１個分の厚みを超える厚みを有する３次元構造を有してもよい。例えば、シート状細胞培養物の垂直断面において、細胞が水平方向に均一に整列することなく、不均一に（例えば、モザイク状に）配置された状態で存在していてもよい。 In the present invention, the term "sheet-like cell culture" refers to a sheet-like cell formed by connecting cells to each other. Cells may be connected to each other directly (including through cellular elements such as adhesion molecules) and/or through intermediaries. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include extracellular matrix. The mediator is preferably of cell origin, in particular of cells that make up the cell culture. The cells are at least physically (mechanically) linked, but may also be functionally linked, eg chemically, electrically. The sheet-like cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminated (multilayered) body, e.g., two layers, three layers, 4 layers, 5 layers, 6 layers, etc.). Moreover, the sheet-like cell culture may have a three-dimensional structure in which the cells do not exhibit a clear layered structure and have a thickness exceeding the thickness of a single cell. For example, in a vertical cross section of a sheet-like cell culture, the cells may be non-uniformly arranged (for example, in a mosaic pattern) without being evenly aligned in the horizontal direction.
　シート状細胞培養物は、好ましくはスキャフォールド（支持体）を含まない。スキャフォールドは、その表面上および／またはその内部に細胞を付着させ、シート状細胞培養物の物理的一体性を維持するために当該技術分野において用いられることがある。本発明のシート状細胞培養物は、かかるスキャフォールドがなくともその物理的一体性を維持することができる。また、本発明のシート状細胞培養物は、フィブリン等を塗布することで、その物理的強度を補強することもできる。 The sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds are sometimes used in the art to attach cells onto and/or into their surfaces and maintain the physical integrity of sheet-like cell cultures. The sheet-like cell culture of the present invention can maintain its physical integrity without such a scaffold. In addition, the sheet-like cell culture of the present invention can be coated with fibrin or the like to reinforce its physical strength.
　シート状細胞培養物を構成する「細胞」は、特に限定されず、例えば、接着細胞（付着性細胞）が挙げられる。接着細胞は、例えば、接着性の体細胞等を含む。体細胞の例としては、例えば、筋芽細胞（例えば、骨格筋芽細胞等）、筋衛星細胞、間葉系幹細胞（例えば、骨髄、脂肪組織、末梢血、皮膚、毛根、筋組織、子宮内膜、胎盤、臍帯血由来のもの等）、心筋細胞、線維芽細胞、心臓幹細胞等の組織幹細胞、胚性幹細胞、ｉＰＳ（induced pluripotent stem）細胞等の多能性幹細胞、滑膜細胞、軟骨細胞、上皮細胞（例えば、口腔粘膜上皮細胞、網膜色素上皮細胞、鼻粘膜上皮細胞等）、内皮細胞（例えば、血管内皮細胞等）、肝細胞（例えば、肝実質細胞等）、膵細胞（例えば、膵島細胞等）、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞等が挙げられる。体細胞は、ｉＰＳ細胞から分化させたもの（ｉＰＳ細胞由来細胞）であってよく、ｉＰＳ細胞由来の心筋細胞、線維芽細胞、筋芽細胞、上皮細胞、内皮細胞、肝細胞、膵細胞、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞、滑膜細胞、軟骨細胞等が挙げられる。 The "cells" that make up the sheet-like cell culture are not particularly limited, and examples include adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells and the like. Examples of somatic cells include myoblasts (e.g., skeletal myoblasts), muscle satellite cells, mesenchymal stem cells (e.g., bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, intrauterine tissue stem cells such as cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, synovial cells, chondrocytes , Epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., hepatocytes, etc.), pancreatic cells (e.g., pancreatic islet cells, etc.), renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells and the like. Somatic cells may be those differentiated from iPS cells (iPS cell-derived cells), iPS cell-derived cardiomyocytes, fibroblasts, myoblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, and the like.
　本発明において、「細胞懸濁液」とは、細胞が懸濁された液体媒体をいう。液体媒体としては、細胞の操作に通常使用する任意の媒体、限定されずに、例えば、生理食塩水、リンゲル液、ハンクス平衡塩液などの平衡塩液、ＰＢＳ（リン酸緩衝生理食塩水）、液体培地などを用いることができる。 In the present invention, "cell suspension" refers to a liquid medium in which cells are suspended. As a liquid medium, any medium commonly used for manipulation of cells, including but not limited to, balanced salt solutions such as saline, Ringer's solution, Hank's balanced salt solution, PBS (phosphate buffered saline), liquid A medium or the like can be used.
　シート状細胞培養物は、既知の任意の方法（例えば、特許文献１、特許文献２、特開２０１０－０８１８２９、特開２０１１－１１０３６８など参照）で製造することができる。シート状細胞培養物の製造方法は、典型的には、実質的に増殖することなくシート状細胞培養物を形成し得る密度で細胞（細胞懸濁液）を培養容器内に播種するステップ、播種した細胞をシート化するステップ、形成されたシート状細胞培養物を培養容器から剥離するステップを含むが、これらに限定されない。これら各ステップは、シート状細胞培養物の製造に適した既知の任意の手法で行うことができる。本発明は、シート状細胞培養物を製造するステップを含んでもよく、その場合、シート状細胞培養物を製造するステップは、上記システムを使用してシート状細胞培養物を製造することを含んでもよい。 A sheet-like cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, JP-A-2010-081829, JP-A-2011-110368, etc.). A method for producing a sheet-like cell culture typically includes the step of seeding cells (cell suspension) in a culture vessel at a density that allows formation of a sheet-like cell culture without substantial proliferation, seeding It includes, but is not limited to, forming a sheet from the cells that have been cultured, and peeling off the formed sheet-like cell culture from the culture vessel. Each of these steps can be performed by any known method suitable for producing a sheet-like cell culture. The present invention may comprise a step of producing a sheet-like cell culture, wherein the step of producing a sheet-like cell culture comprises producing a sheet-like cell culture using the above system. good.
　本発明において、「細胞懸濁液を播種」するとは、細胞懸濁液を培養容器内に播種することをいい、「シート化培養」とは、培養容器内に播種した細胞懸濁液（細胞）をインキュベートすることにより、シート状細胞培養物を形成することをいう。シート化培養においては、培養容器内に播種された細胞懸濁液中の細胞は、播種直後には液体媒体中に浮遊しており、時間が経つにつれて培養容器の底面に徐々に沈降していく。そして、細胞は培養容器の底面に接着して、シート状細胞培養物の一部を構成し、さらに細胞同士が接着することでシート化が進行してシート状細胞培養物が形成される。本発明において、液体媒体中に浮遊している細胞を「非接着細胞」、培養容器の底面に接着している細胞を「接着細胞」という場合がある。 In the present invention, "seeding a cell suspension" refers to seeding a cell suspension in a culture vessel, and "sheet culture" refers to a cell suspension seeded in a culture vessel (cell ) to form a sheet-like cell culture. In sheet culture, cells in a cell suspension seeded in a culture vessel float in a liquid medium immediately after seeding, and gradually settle to the bottom of the culture vessel over time. . Then, the cells adhere to the bottom surface of the culture vessel to form a part of the sheet-like cell culture, and the cells adhere to each other to form a sheet, thereby forming a sheet-like cell culture. In the present invention, cells floating in a liquid medium are sometimes referred to as "non-adherent cells", and cells adhering to the bottom surface of a culture vessel are sometimes referred to as "adherent cells".
　本発明において、「細胞の分布」とは、細胞懸濁液中における細胞の分布をいう。細胞懸濁液中の細胞は、播種直後には球状で液体媒体中に浮遊し、細胞同士の距離が離れた状態（細胞同士が接着していない状態）で分布するため、これをシート状細胞培養物の未形成状態を評価するための指標とすることができる。次に、細胞は徐々に沈降していき、培養容器の底面に接着するため、培養容器の底面より上の部分における細胞の分布は減少すると同時に、底面における細胞の分布は増加するため、これらの変化をシート状細胞培養物の形成開始状態を評価するための指標とすることができる。 In the present invention, "cell distribution" refers to the distribution of cells in a cell suspension. Cells in a cell suspension are spherical immediately after being seeded and float in a liquid medium, and are distributed in a state in which the cells are separated from each other (the cells do not adhere to each other). It can be used as an index for evaluating the unformed state of the culture. Next, since the cells gradually settle and adhere to the bottom surface of the culture vessel, the distribution of cells on the part above the bottom surface of the culture vessel decreases, while the distribution of cells on the bottom surface increases. The change can be used as an index for evaluating the formation start state of the sheet-like cell culture.
　培養容器の底面に接着した細胞の分布が小さい（沈降した細胞の数が少ない）ときは、１細胞当たりの接着できる面積が大きく、各細胞は接着後底面上で広がった形状に変化していくが、細胞の沈降が進み、培養容器の底面に接着した細胞の分布が大きくなる（沈降した細胞の数が増加する）につれ、１細胞当たりの接着できる面積は小さくなる。また、各細胞は隣接する他の細胞と接着し始め、細胞間の距離が近くなる。したがって、これらの変化をシート状細胞培養物の形成進行状態を評価するための指標とすることができる。 When the distribution of cells adhered to the bottom surface of the culture vessel is small (the number of settled cells is small), the area that can be adhered per cell is large, and each cell changes its shape to spread on the bottom surface after adhesion. However, as the sedimentation of cells progresses and the distribution of cells adhered to the bottom surface of the culture vessel increases (the number of sedimented cells increases), the area per cell that can adhere becomes smaller. Also, each cell begins to adhere to other neighboring cells, bringing the cells closer together. Therefore, these changes can be used as indicators for evaluating the progress of sheet-like cell culture formation.
　さらに細胞の沈降が進み、細胞が培養容器の底面全体で飽和状態となることで、あるタイミングで細胞の変化が急激に低下して一定になり、１細胞当たりの接着面積がさらに小さくなり、シート化培養が完了する。したがって、これらをシート状細胞培養物の完成状態を判定するための指標とすることができる。細胞は、常に細胞骨格を変化させて形状変化や移動・収縮する性質があるが、形状変化や移動するスペースはなく、収縮の動きに応答して、各細胞に強固に接着している隣接する細胞が引っ張られるため、ある時点ある領域でシート状細胞培養物が収縮すると、細胞間接着より弱くシャーレ周囲と接着していた周縁部から自然剥離が始まる。シート状細胞培養物の接着した部分の細胞に対して、剥離した部分の細胞は浮遊しており、剥離した細胞は徐々に元の形状（球状）に戻ろうとする性質があることから、さらに収縮が進んでいるため、これらをシート状細胞培養物の剥離状態を評価するための指標とすることができる。 As the sedimentation of the cells progresses further and the cells reach a state of saturation on the entire bottom surface of the culture vessel, at a certain timing the change in the cells suddenly decreases and becomes constant, the adhesion area per cell becomes even smaller, and the sheet becomes Cultivation is completed. Therefore, these can be used as indicators for determining the completed state of the sheet-like cell culture. Cells have the property of constantly changing their cytoskeleton to change shape, migrate, and contract. Since the cells are pulled, when the sheet-like cell culture shrinks in a certain area at a certain point in time, natural detachment starts from the periphery, which was weaker than the cell-to-cell adhesion and adhered to the periphery of the petri dish. Compared to the cells in the adhered part of the sheet-like cell culture, the cells in the detached part are floating, and the detached cells have the property of gradually returning to their original shape (spherical), so they shrink further. These can be used as indicators for evaluating the detachment state of sheet-like cell cultures.
　本発明の解析部は、細胞懸濁液中の細胞の分布から、細胞懸濁液中の細胞の密度分布を算出することができる。例えば、解析部は、細胞懸濁液中の細胞の分布（細胞の動き）を立体的に捉え、細胞の密度分布として算出することで、シート状細胞培養物のシート化培養の進行を解析することができる。すなわち、細胞が細胞懸濁液中で浮遊、沈降、接着、剥離することと、シート状細胞培養物のシート化培養の進行とは対応するため、細胞懸濁液中の各部位における細胞の分布は、シート化培養の指標として使用することができる。細胞懸濁液中の各部位は、培養容器の底面、底面より上、底面の中心、底面の周縁など、細胞懸濁液を立体的に捉えた場合の例えばｘ、ｙ、ｚ座標系で表すことができる。 The analysis unit of the present invention can calculate the density distribution of cells in the cell suspension from the distribution of cells in the cell suspension. For example, the analysis unit three-dimensionally captures the cell distribution (cell movement) in the cell suspension and calculates the density distribution of the cells to analyze the progress of sheet-like cell culture. be able to. That is, since the floating, sedimentation, adhesion, and detachment of cells in a cell suspension correspond to the progress of sheet-like cell culture in sheet-like cell culture, the distribution of cells at each site in the cell suspension can be used as an index for sheet culture. Each part in the cell suspension is represented by an x, y, z coordinate system when the cell suspension is three-dimensionally captured, such as the bottom of the culture vessel, above the bottom, the center of the bottom, and the periphery of the bottom. be able to.
　細胞懸濁液中の細胞の分布が、培養容器の底面において不均一な場合、製造されるシート状細胞培養物にムラが生じる。ここでいうムラとはシート状細胞培養物において厚みの違う部分（厚みが不均一な部分）があることを意味し、このような部分がシート状細胞培養物に散在しているとシート剥離から使用までの間に厚みの不均一（ムラ）のためにシート状細胞培養物に破れや破損が生じる可能性がある。したがって、シート状細胞培養物を製造するにあたり、培養容器の底面の細胞の分布の均一性を測定して、このようなムラの発生を避ける必要がある。 If the distribution of cells in the cell suspension is uneven on the bottom of the culture vessel, the produced sheet-like cell culture will be uneven. The unevenness referred to here means that the sheet-shaped cell culture has portions with different thicknesses (non-uniform thickness portions). The sheet-like cell culture may be torn or damaged due to non-uniform thickness (unevenness) before use. Therefore, in producing a sheet-like cell culture, it is necessary to measure the uniformity of cell distribution on the bottom surface of the culture vessel to avoid such unevenness.
　本発明において、「シート化状態」とは、シート状細胞培養物の形成度合い、および／またはシート状細胞培養物の剥離度合いを含む、シート状細胞培養物の状態を定量的に示すものであり、細胞懸濁液中における細胞の分布から一義的に決定され得る。「シート状細胞培養物の形成度合い」としては、これに限定するものではないが、例えば、シート状細胞培養物の完成状態を１００％としたときの完成度を百分率で表したものでもよいし、設定値以上の完成度のものを「合格」とするものであってもよい。すなわち、シート状細胞培養物の形成度合いは、測定された細胞懸濁液中の細胞の浮遊、沈降、接着に伴う細胞の分布から算出することで数値化できる。 In the present invention, the “sheet state” quantitatively indicates the state of the sheet-like cell culture, including the degree of formation of the sheet-like cell culture and/or the degree of detachment of the sheet-like cell culture. , can be uniquely determined from the distribution of the cells in the cell suspension. The "formation degree of the sheet-like cell culture" is not limited to this, but may be, for example, a degree of completion expressed as a percentage when the completed state of the sheet-like cell culture is 100%. , the degree of perfection equal to or higher than the set value may be set as "accepted". That is, the degree of sheet-like cell culture formation can be quantified by calculating from the distribution of cells associated with floating, sedimentation, and adhesion of cells in the measured cell suspension.
　「シート状細胞培養物の剥離度合い」は、例えば、シート状細胞培養物の培養容器の底面に接着している部分と、剥離している部分との面積比率を測定することで数値化できる。また、シート状細胞培養物の剥離度合いは、例えば、完成状態が１００％であるシート状細胞培養物全体が、培養容器の底面に接着している状態を０％（剥離なし）、一部が自然剥離してその面積の１％が液体媒体中にある状態を１％（１％剥離）とすることで、数値化できる。また、完成状態が１００％に満たない未完成のシート状細胞培養物でも、自然剥離した部分の底面に対する面積を算出することで、剥離度合いを算出することができる。 The "degree of detachment of the sheet-like cell culture" can be quantified, for example, by measuring the area ratio of the part of the sheet-like cell culture adhered to the bottom of the culture vessel and the detached part. In addition, the degree of detachment of the sheet-like cell culture is, for example, 0% (no detachment) when the entire sheet-like cell culture in a completed state of 100% is adhered to the bottom surface of the culture vessel, and a part is A state in which 1% of the area is in the liquid medium due to natural peeling is defined as 1% (1% peeling), which can be quantified. In addition, even in an incomplete sheet-like cell culture whose completed state is less than 100%, the degree of detachment can be calculated by calculating the area of the naturally detached portion with respect to the bottom surface.
　シート状細胞培養物の自然剥離が進行すると、細胞が収縮して剥離する部分が大きくなり、そのまま放置するとシート状細胞培養物が丸まっていき、出荷に相応しくない状態になることがある。また、シート状細胞培養物が未完成のままで細胞の自然剥離が進行すると、シート状細胞培養物が上手く形成されず、出荷に相応しくない状態になることがある。したがって、シート状細胞培養物の剥離度合いが設定値以上になったときは、剥離処理（強制剥離）を行うことが好ましい。かかる設定値は、例えば、剥離度合いが５％のときなど、自由に設定できる。本発明においては、シート状細胞培養物のシート化状態の変化から、完成のタイミング予測、自然剥離のタイミング予測、剥離処理（強制剥離）のタイミング予測などを行うこともできる。 As the sheet-shaped cell culture naturally detaches, the cells shrink and the detached part becomes larger. If left as it is, the sheet-shaped cell culture may curl up and become unsuitable for shipment. In addition, if the spontaneous detachment of the cells progresses while the sheet-like cell culture is incomplete, the sheet-like cell culture may not be formed well and may be unsuitable for shipment. Therefore, when the degree of detachment of the sheet-like cell culture reaches or exceeds a set value, detachment treatment (forced detachment) is preferably performed. Such a set value can be freely set, for example, when the degree of peeling is 5%. In the present invention, it is also possible to predict the timing of completion, the timing of spontaneous detachment, the timing of detachment treatment (forced detachment), and the like, based on changes in the state of sheet-like cell culture.
　シート化状態は、細胞懸濁液中の細胞の浮遊、沈降、接着、剥離という、一連の細胞の分布の状態を経時的に測定することで、数値的な変化を算出することができる。例えば、シート状細胞培養物の完成状態を１００％とし、剥離度合いを１％とすることで、シート化状態が１０１％進んでいると定義することもできる。そして、例えば、シート化状態が、第１の設定値（例えば、１００％以上）を超えたときに、シート状細胞培養物が完成したと判定し、第２の設定値（例えば、１０１％以上）を超えたときに、シート状細胞培養物の剥離処理が必要であると判定することもできる。また、シート化培養中の（完成前のシート状細胞培養物の）細胞の剥離を異常剥離として判定することもできる。 For the sheet state, numerical changes can be calculated by measuring a series of cell distribution states such as floating, sedimentation, adhesion, and detachment of cells in a cell suspension over time. For example, by setting the completed state of the sheet-shaped cell culture to 100% and the degree of peeling to 1%, it can be defined that the sheet-formed state has progressed 101%. Then, for example, when the sheet-formed state exceeds a first set value (e.g., 100% or more), it is determined that the sheet-like cell culture is completed, and the second set value (e.g., 101% or more) ), it can be determined that the sheet-like cell culture needs to be peeled off. In addition, detachment of cells during sheet culture (of a sheet-like cell culture before completion) can also be determined as abnormal detachment.
　すなわち、シート化状態が、第１の設定値（例えば、１００％以上）を超える前で（例えば、９０％のときに）、シート状細胞培養物の自然剥離が始まった場合は、シート化状態が９０％で剥離が起こった不良品と判定することもできる。さらに、シート化状態の数値変化（例えば、完成度の変化）を解析することで、シート状細胞培養物が完成するタイミングや、自然剥離が始まるタイミングを予測することもできる。これらの予測は、作業者が行ってもよいが、予め用意した検量線と比較することで、精度を高めることができる。機械学習などで生成した学習済みモデルを用いることで、さらに精度を高めることもできる。 That is, before the sheet state exceeds a first set value (e.g., 100% or more) (e.g., when it is 90%), if the sheet-like cell culture begins to spontaneously detach, the sheet state It can also be determined as a defective product in which peeling occurred at 90%. Furthermore, by analyzing numerical changes in the sheet-formed state (for example, changes in the degree of completion), it is possible to predict the timing at which the sheet-shaped cell culture is completed and the timing at which spontaneous detachment begins. These predictions may be made by an operator, but the precision can be improved by comparing with a calibration curve prepared in advance. Accuracy can be further improved by using a trained model generated by machine learning or the like.
　本発明において、「測定部」とは、細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得する部分をいう。測定部は、典型的には光検出器を含み、光検出器は、限定されず、例えば光電センサ、ファイバセンサ、レーザセンサ、カラーセンサなど、細胞懸濁液を播種した培養容器内の細胞の分布を光検出で測定できる装置を含むことができる。 In the present invention, the "measurement part" refers to a part that acquires information on the distribution of cells in the culture vessel in which the cell suspension is seeded. The measurement unit typically includes a photodetector, and the photodetector is not limited and includes, for example, a photoelectric sensor, a fiber sensor, a laser sensor, a color sensor, and the like. A device can be included that can measure the distribution with photodetection.
　シート化培養においては、細胞懸濁液を培養容器内に播種し、一般的に、温度を３７℃に維持しながらインキュベートすることで、シート状細胞培養物を形成する。この際に、培養容器内の液体媒体が気化して湿気が発生し、かかる湿気が測定部の検知を（例えば、レンズを曇らせることで）妨げる場合がある。また、コンタミネーションのリスクを最小限にするために、培養容器には、一般的に蓋をしてインキュベートを行うが、湿気が測定部の検知を（例えば、蓋を曇らせることで）妨げる場合がある。このような問題を解決するために、本発明の測定部は、一態様において、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するように構成することができる。 In sheet-like culture, a cell suspension is seeded in a culture vessel and generally incubated while maintaining the temperature at 37°C to form a sheet-like cell culture. At this time, the liquid medium in the culture vessel evaporates to generate moisture, which may interfere with the detection of the measurement unit (for example, by fogging up the lens). Also, to minimize the risk of contamination, culture vessels are typically incubated with a lid, but moisture can interfere with detection of the assay (e.g., by fogging up the lid). be. In order to solve such problems, in one aspect, the measurement unit of the present invention measures cells in the culture vessel from the bottom direction and / or side direction in contact with the cell suspension of the culture vessel. can be configured as
　本発明において、「培養容器の細胞懸濁液と接触している部分から細胞を測定する」とは、培養容器内の細胞懸濁液に存在する細胞を細胞懸濁液の液面からではなく、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定することをいう。上記のような湿気は、細胞懸濁液の液面から発生するため、本発明における測定部は、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定することで、液面から発生する湿気の影響を受けないようにすることができる。また、これにより観察時に細胞懸濁液の液面で起こりうる、光の屈折、反射、回折などの影響を最小限に抑えることができる。測定部を培養容器の側面や底面に密着させて（測定部を培養容器から離間させずに）、培養容器内の細胞を測定することもできる。 In the present invention, "measure cells from the portion of the culture vessel that is in contact with the cell suspension" means that the cells present in the cell suspension in the culture vessel are measured from the liquid surface of the cell suspension. , refers to measuring the cells in the culture vessel from the bottom direction and/or the side direction in contact with the cell suspension of the culture vessel. Since the moisture as described above is generated from the liquid surface of the cell suspension, the measuring unit in the present invention measures the humidity inside the culture vessel from the bottom direction and/or the side direction, which is in contact with the cell suspension of the culture vessel. By measuring the cells of the liquid, it is possible to avoid the influence of moisture generated from the liquid surface. In addition, it is possible to minimize the effects of light refraction, reflection, and diffraction that may occur on the liquid surface of the cell suspension during observation. Cells in the culture vessel can also be measured by attaching the measurement section to the side or bottom of the culture vessel (without separating the measurement section from the culture vessel).
　測定部として光検出器を使用する場合は、例えば、投受光器で、細胞からの反射光を測定する方法（反射型）、投光器と受光器とで細胞を挟むように配置し、細胞を透過した透過光を測定する方法（透過型）、投受光器とリフレクタとで細胞を挟むように配置して、反射光を測定する方法（回帰反射型）など、様々な方法や配置で、細胞を測定することができる。上記のような、細胞懸濁液中の細胞の浮遊、沈降、接着、剥離という、細胞の分布の一連の変化を正確に測定するために、測定部の少なくとも測定側（投受光器、受光器など）を培養容器の底面側および／または側面側に配置することができる。また、光は発散するため、測定部の非測定側（投光器など）はどこに配置してもよいが、好ましくは湿気の影響を受けにくい培養容器の側面側および／または底面側に配置する。 When using a photodetector as the measurement part, for example, a method of measuring the light reflected from the cell with a light emitter and receiver (reflection type), or placing the light emitter and light receiver so that the cell is sandwiched between them allows the cell to pass through. Cells can be measured by various methods and arrangements, such as a method of measuring transmitted light (transmissive type), and a method of measuring reflected light by placing a cell between a light emitter and a reflector (retroreflective type). can be measured. In order to accurately measure a series of changes in cell distribution such as floating, sedimentation, adhesion, and detachment of cells in the cell suspension, at least the measurement side of the measurement unit (projector and receiver, receiver etc.) can be placed on the bottom and/or side of the culture vessel. In addition, since the light diverges, the non-measurement side of the measurement unit (projector, etc.) can be placed anywhere, but is preferably placed on the side and/or bottom side of the culture vessel, which is less susceptible to moisture.
　特許文献１、特許文献２などの従来のシステムにおいては、撮像部で撮影した培養容器底面の画像データを解析して、接着細胞率や非接着細胞率を細胞数の計数から算出するが、かかる方法は画像解析のための解析コスト（装置自体の費用、解析に使用するソフトウェアの費用、熟練者の費用など）が高い。また、細胞懸濁液中で浮遊、沈降、接着しながら移動する各細胞と光学顕微鏡との距離は変化するため、各細胞に光学顕微鏡の焦点を合わせない限り、細胞の計数を画像解析による正確に行うことは難しく、高度な技術が必要とされる。したがって、従来のシステムでは通常、培養容器底面に焦点を合わせた細胞の情報（細胞の２次元画像）を解析するように構成されている。 In conventional systems such as Patent Document 1 and Patent Document 2, the image data of the bottom surface of the culture vessel captured by the imaging unit is analyzed, and the adherent cell rate and non-adherent cell rate are calculated from the number of cells. The method has a high analysis cost for image analysis (cost of the device itself, cost of software used for analysis, cost of skilled personnel, etc.). In addition, since the distance between each cell floating, sedimenting, and adhering in the cell suspension and the optical microscope changes, unless the optical microscope is focused on each cell, it is difficult to accurately count cells by image analysis. However, it is difficult to do so and requires advanced technology. Therefore, conventional systems are generally configured to analyze cell information (two-dimensional image of cells) focused on the bottom surface of the culture vessel.
　これに対して、本発明のシステムは、測定部を培養容器の細胞懸濁液と接触している底面方向および／または側面方向に配置するという簡単な機構を採用することで、シート状細胞培養物のシート化状態を正確に評価できる。例えば、培養容器の側面側から細胞を測定することで、細胞の垂直方向（ｚ軸方向）の移動（浮遊、沈降、剥離など）や分布を湿気の影響を受けずに測定することができる。また、培養容器の底面側から細胞を測定することで、細胞の水平方向（ｘ軸方向、ｙ軸方向）の移動（接着、剥離など）や分布を湿気の影響を受けずに測定することができる。また、測定部は、培養容器の側面側から底面方向、および／または底面側から側面方向のように、培養容器を立体的に斜めに横断するように測定することで、細胞の垂直方向（ｚ軸方向）と水平方向（ｘ軸方向、ｙ軸方向）の移動とを同時に測定することもできる。 In contrast, the system of the present invention adopts a simple mechanism in which the measurement unit is arranged in the direction of the bottom surface and/or the side surface in contact with the cell suspension of the culture vessel, thereby enabling sheet-like cell culture. It is possible to accurately evaluate the sheeting state of an object. For example, by measuring cells from the side of the culture vessel, it is possible to measure the movement (floating, sedimentation, detachment, etc.) and distribution of cells in the vertical direction (z-axis direction) without being affected by humidity. In addition, by measuring cells from the bottom side of the culture vessel, it is possible to measure the movement (adhesion, detachment, etc.) and distribution of cells in the horizontal direction (x-axis direction, y-axis direction) without being affected by humidity. can. In addition, the measurement unit performs measurement so as to three-dimensionally cross the culture vessel diagonally, such as from the side of the culture vessel to the bottom direction and/or from the bottom side to the side direction, so that the vertical direction (z Axial) and horizontal (x-axis, y-axis) movements can also be measured simultaneously.
　測定部は、培養容器の側面側および／または底面側に少なくとも１つ、好ましくは複数配置することで、細胞懸濁液中の細胞の垂直方向および／または水平方向の移動を各部位毎に立体的に測定できるため、細胞の分布を測定するのに有利である。すなわち、従来のシステムでは、細胞を培養容器底面の面積当たりの２次元情報として測定するのに対して、本発明のシステムでは、細胞を細胞懸濁液の体積当たりの３次元情報として測定できるため、シート化状態をより正確に評価できる。また、測定部として光検出器を使用する場合は、細胞懸濁液の細胞の分布の情報を光の透過率や反射率の情報として測定できるため、画像解析のような高度な技術を必要とせず、解析コストを低く抑えられる。 By arranging at least one, preferably a plurality of measurement units on the side surface and/or the bottom surface of the culture vessel, the movement of cells in the cell suspension in the vertical direction and/or the horizontal direction can be measured three-dimensionally for each region. It is advantageous for measuring the distribution of cells because it can be measured in a specific manner. That is, in the conventional system, cells are measured as two-dimensional information per area of the bottom surface of the culture vessel, whereas in the system of the present invention, cells can be measured as three-dimensional information per volume of the cell suspension. , the sheeting state can be evaluated more accurately. In addition, when a photodetector is used as the measurement unit, information on the distribution of cells in the cell suspension can be measured as information on the transmittance and reflectance of light, so advanced techniques such as image analysis are not required. analysis cost can be kept low.
　本発明において、「解析部」とは、測定部により取得された情報を解析してシート状細胞培養物の形成度合いおよび／または剥離度合いを算出する部分をいう。解析部は、測定部により取得された情報を解析するためのプロセッサなどを含むことができる。測定部により取得された情報は、細胞の分布に関する情報を含み、かかる情報は、例えば、細胞懸濁液中の各部位における細胞に対する光の透過率や反射率の情報を含むことができる。すなわち、細胞懸濁液中で細胞が多く分布している部位と、少なく分布している部位とでは、光の透過率や反射率が異なり、これらの情報を解析して細胞の密度分布を算出し、シート化状態を評価することができる。また、測定部からの情報を経時的に解析し、その経時的変化から、シート状細胞培養物のシート化状態の変化をグラフ化することもできる。 In the present invention, the "analysis part" refers to a part that analyzes the information acquired by the measurement part and calculates the degree of formation and/or the degree of detachment of the sheet-like cell culture. The analysis unit can include a processor or the like for analyzing information obtained by the measurement unit. The information acquired by the measurement unit includes information on cell distribution, and such information can include, for example, information on light transmittance and reflectance for cells at each site in the cell suspension. In other words, the light transmittance and reflectance are different between areas where a large number of cells are distributed and areas where a small number of cells are distributed in the cell suspension, and the cell density distribution is calculated by analyzing these information. It is possible to evaluate the sheeting state. It is also possible to analyze the information from the measurement unit over time, and graph the changes in the sheet-formed state of the sheet-like cell culture based on the change over time.
　解析部は、シート状細胞培養物のシート化状態（形成度合いおよび／または剥離度合い）を数値化し、かかる算出値をリアルタイムで出力することができる。また、算出値が第１の設定値以上である場合に、シート状細胞培養物が完成したと判定し、算出値が第２の設定値以上である場合に、シート状細胞培養物が自然剥離したと判定することもできる。すなわち、解析部は、例えば、算出や判定に関する情報をリアルタイムで出力することができる。算出や判定に関する情報は、完成のタイミング、自然剥離のタイミング、剥離処理（強制剥離）のタイミングに関する予測（時刻やカウントダウンなど）を含むこともできる。 The analysis unit can quantify the sheet-forming state (degree of formation and/or degree of detachment) of the sheet-shaped cell culture and output such calculated values in real time. Further, when the calculated value is the first set value or more, it is determined that the sheet-like cell culture is completed, and when the calculated value is the second set value or more, the sheet-like cell culture is naturally exfoliated. It can also be determined that That is, the analysis unit can output, for example, information regarding calculation and determination in real time. Information related to calculation and determination can also include predictions (time, countdown, etc.) regarding the timing of completion, the timing of natural peeling, and the timing of peeling processing (forced peeling).
　形成および／または剥離の判定は、作業者が、シート化状態の数値に基づいて行ってもよいし、システムが、かかる数値と設定値とを比較することで行ってもよい。解析部による判定は、予め用意した検量線や学習済みモデルを用いて行うこともできる。学習済みモデルは、例えば、熟練者が、細胞を計数したり、画像解析したりして導き出したシート化状態の生データから、欠測値や外れ値の除去、正解データの抽出を行うことで、学習用データセットを生成し、学習用データセットを使った学習の結果として、学習済パラメータを生成する。正解データは、熟練者の持つ、細胞の均一性、厚みのムラ、シート化状態、剥離などに関する経験に基づいた知識を含めることができる。 Formation and/or peeling may be determined by the operator based on the numerical value of the sheeting state, or by the system comparing the numerical value with the set value. The determination by the analysis unit can also be performed using a calibration curve prepared in advance or a learned model. A trained model is created by removing missing values and outliers and extracting correct data from raw data in a sheet state derived from cell counting and image analysis by an expert, for example. , generate a training data set, and generate learned parameters as a result of learning using the training data set. The correct answer data can include knowledge based on experiences of experts regarding cell uniformity, thickness unevenness, sheet state, peeling, and the like.
　そして、学習済みパラメータが組み込まれた推論プログラム、すなわち、組み込まれた学習済みパラメータを適用することにより、入力に対して一定の結果が出力されることを可能にするプログラムとして、学習済みモデルを生成することができる。さらに、生データとして、光検出器で測定した情報を、画像解析により得られた情報に追加的に読み込ませることによって、光検出器で測定した情報に対して好適に使用できる学習済みモデルを生成することができる。これにより、光検出器で測定した情報を解析してシート化培養の様々なパターンを認識することができる。当業者であれば、種々の学習データ、学習方法、学習モデルを組み合わせて、最適な学習モデルを生成することができる。 Then, a trained model is generated as an inference program that incorporates the learned parameters, i.e., a program that can output a certain result for the input by applying the built-in learned parameters. can do. Furthermore, by additionally loading the information obtained by image analysis with the information measured by the photodetector as raw data, a trained model that can be suitably used for the information measured by the photodetector is generated. can do. As a result, various patterns of sheet culture can be recognized by analyzing the information measured by the photodetector. A person skilled in the art can combine various learning data, learning methods, and learning models to generate an optimal learning model.
　本発明のシステムは、少なくとも測定部を含むが、さらに、解析部、記憶部、入力部、出力部などを含んでもよい。記憶部は、測定部により取得された情報、解析部により解析された情報、システムを作動させるためのプログラム等を保存する部分であり、種々の電子記憶媒体、例えば、半導体メモリ、ハードディスク、クラウドストレージ等を含むことができる。入力部は、システムの操作者、または外部のシステムが、測定パラメータやプログラムを入力する部分であり、種々の入力インターフェース、例えば、電気や光等の信号を他のシステムから受け取る手段（電線、光ファイバー、コネクタ、無線通信装置等）、ボタン、キーボード、タッチパネル等を含むことができる。 The system of the present invention includes at least a measurement unit, but may further include an analysis unit, a storage unit, an input unit, an output unit, and the like. The storage unit is a part that stores information acquired by the measurement unit, information analyzed by the analysis unit, programs for operating the system, etc. Various electronic storage media such as semiconductor memory, hard disk, cloud storage etc. The input part is a part where the operator of the system or an external system inputs measurement parameters and programs, and various input interfaces, for example, means for receiving signals such as electricity and light from other systems , connectors, wireless communication devices, etc.), buttons, keyboards, touch panels, and the like.
　出力部は、測定部により取得された情報、解析部により解析された情報などに基づき、所定の信号や情報を出力する部分であり、種々の出力インターフェース、例えば、電気や光、情報等を出力する手段（電線、光ファイバー、コネクタ、無線通信装置等）、ディスプレイ、タッチパネル、プリンタ、パトランプ、ブザー、音声合成装置等を含むことができる。入力部および出力部を一体化して、汎用コンピュータやスマート端末（スマートフォン、タブレット端末）として利用してもよい。本発明のシステムは、汎用コンピュータのためのソフトウェアや、スマート端末のためのアプリケーションを含んでもよく、測定部により取得された情報、解析部により解析された情報などをメールやアプリプッシュ通知で、使用者に送信するように構成することもできる。 The output section is a section that outputs predetermined signals and information based on the information acquired by the measurement section and the information analyzed by the analysis section, and outputs various output interfaces, such as electricity, light, and information. means (electric wire, optical fiber, connector, wireless communication device, etc.), display, touch panel, printer, patrol lamp, buzzer, voice synthesizer, etc. can be included. An input unit and an output unit may be integrated to be used as a general-purpose computer or a smart terminal (smartphone, tablet terminal). The system of the present invention may include software for general-purpose computers and applications for smart terminals, and the information acquired by the measurement unit, the information analyzed by the analysis unit, etc. are used by email or application push notification. It can also be configured to send to
　本発明のシステムは、さらに、シート状細胞培養物を剥離するための剥離部を含んでもよい。すなわち、上記のように、シート状細胞培養物（完成、未完成を含む）の剥離は、出荷に相応しくない状態を招くため、シート状細胞培養物の剥離度合いが設定値以上になったときに、剥離部で、剥離処理（強制処理）を行うように構成することもできる。かかる剥離部としては、例えば、液体を振動させる手段（特開２０１６－５２２６９を参照）を用いることができる。また、剥離部は、剥離したシート状細胞培養物を伸展させる手段（特開２０１６－５２２７０を参照）をさらに含むこともできる。剥離部は、これらに限定されず、種々の公知の手段を用いたり、組み合わせたりすることができる。 The system of the present invention may further include a peeling section for peeling the sheet-like cell culture. That is, as described above, detachment of the sheet-like cell culture (including completed and unfinished) results in a state unsuitable for shipment. , the peeling unit may be configured to perform peeling processing (forced processing). As such a separation unit, for example, means for vibrating liquid (see Japanese Patent Laid-Open No. 2016-52269) can be used. In addition, the peeling section can further include a means for spreading the peeled sheet-like cell culture (see Japanese Patent Application Laid-Open No. 2016-52270). The peeling portion is not limited to these, and various known means can be used or combined.
（シート化状態を評価するためのシステム）
　以下に、本発明のシステムを図面を参照してより詳細に説明するが、これは本発明の特定の具体例を示すものであり、これに限定されるものではない。本発明のシステムは、少なくとも測定部を含むが、さらに、解析部、記憶部、入力部、出力部などを含んでもよい。 (System for evaluating the state of sheeting)
In the following, the system of the invention will be described in more detail with reference to the drawings, which illustrate specific embodiments of the invention and are not intended to be limiting thereof. The system of the present invention includes at least a measurement section, and may further include an analysis section, a storage section, an input section, an output section, and the like.
　図１は、本発明のシステムの一態様における概念図を示す。
　図１Ａに示されるように、本発明のシステムは、測定部１を含み、測定部１は、細胞懸濁液Ｌを播種した培養容器Ｃの細胞懸濁液Ｌと接触している部分（側面）から、細胞の垂直方向（ｚ軸方向）および／または水平方向（ｘ軸方向、ｙ軸方向）の移動を測定することができる。また、図１Ｂに示されるように、測定部１は、例えば、培養容器Ｃの底面に１つ、培養容器Ｃの側面を取り囲むように６つ配置することもできる。これにより、細胞懸濁液中の各部位における細胞の分布をより正確に測定することができる。細胞の垂直方向（ｚ軸方向）の移動や分布を測定することで、細胞の浮遊、沈降、剥離などの状態や変化を解析することができる。 FIG. 1 shows a conceptual diagram of one embodiment of the system of the present invention.
As shown in FIG. 1A, the system of the present invention includes a measurement unit 1, and the measurement unit 1 is a portion (side surface) in contact with the cell suspension L of the culture vessel C seeded with the cell suspension L. ), vertical (z-axis) and/or horizontal (x-axis, y-axis) migration of cells can be measured. Further, as shown in FIG. 1B, for example, one measurement unit 1 can be arranged on the bottom surface of the culture container C, and six measurement units can be arranged so as to surround the side surface of the culture container C. As shown in FIG. This makes it possible to more accurately measure the cell distribution at each site in the cell suspension. By measuring the movement and distribution of cells in the vertical direction (z-axis direction), it is possible to analyze states and changes such as cell suspension, sedimentation, and detachment.
　図２は、シート状細胞培養物の周縁部の剥離を検出する様子を示す説明図である。測定部１は投光器（光源）１ａ又は受光器１ｂである。図２の例では、投光器１ａは培養容器の底面に、受光器１ｂは培養容器の側面に配置されている。なお、投光器１ａ及び受光器１ｂの対応関係は逆でもよい。図２上側に示すように、シート状細胞培養物Ｓの剥離が開始していない場合、投光器１ａからシート状細胞培養物Ｓの培養面に対して斜めに照射された光はシート状細胞培養物Ｓの周縁部に遮られ、受光器１ｂで検出されない。一方、図２下側に示すように、シート状細胞培養物Ｓの剥離が周縁部から開始した場合、投光器１ａから照射された光は剥離箇所を通過し、受光器１ｂで検出される。 FIG. 2 is an explanatory diagram showing how the detachment of the peripheral portion of the sheet-shaped cell culture is detected. The measurement unit 1 is a light projector (light source) 1a or a light receiver 1b. In the example of FIG. 2, the light projector 1a is arranged on the bottom surface of the culture container, and the light receiver 1b is arranged on the side surface of the culture container. Note that the correspondence relationship between the light projector 1a and the light receiver 1b may be reversed. As shown in the upper part of FIG. 2, when the peeling of the sheet-like cell culture S has not started, the light emitted obliquely from the light projector 1a to the culture surface of the sheet-like cell culture S is emitted from the sheet-like cell culture. It is blocked by the periphery of S and is not detected by the photodetector 1b. On the other hand, as shown in the lower part of FIG. 2, when the peeling of the sheet-like cell culture S starts from the periphery, the light emitted from the light projector 1a passes through the peeled portion and is detected by the light receiver 1b.
　細胞の水平方向（ｘ軸方向、ｙ軸方向）の移動や分布を測定することで、細胞の接着、剥離などの状態や変化を解析することができる。培養容器Ｃの側面側から底面方向、および／または底面側から側面方向のように、培養容器Ｃを立体的に斜めに横断するように測定することで、細胞の垂直方向（ｚ軸方向）と水平方向（ｘ軸方向、ｙ軸方向）の移動とを同時に測定することもできる。培養容器底面の周縁部を側面側の測定器と、底面側の測定器とで挟むように測定することで、シート状細胞培養物の周縁部の剥離を好適に測定することができる。 By measuring the movement and distribution of cells in the horizontal direction (x-axis direction, y-axis direction), it is possible to analyze the state and changes such as cell adhesion and detachment. By measuring diagonally crossing the culture vessel C in three dimensions, such as from the side to the bottom of the culture vessel C and/or from the bottom to the side, the vertical direction (z-axis direction) of the cells and the Movement in the horizontal direction (x-axis direction, y-axis direction) can also be measured at the same time. Detachment of the peripheral edge of the sheet-like cell culture can be preferably measured by sandwiching the peripheral edge of the bottom surface of the culture vessel between the measuring instrument on the side surface and the measuring instrument on the bottom side.
　図３は、本発明のシステム１００の構成例を示す模式図である。システム１００は、測定部１、端末２を含む。 FIG. 3 is a schematic diagram showing a configuration example of the system 100 of the present invention. A system 100 includes a measurement unit 1 and a terminal 2 .
　端末２は、種々の情報処理が可能な情報処理装置であり、例えばタブレット端末である。タブレット端末は、自装置にインストールされたプログラムを実行することにより、上述の解析部、記憶部、入力部、出力部として機能する。 The terminal 2 is an information processing device capable of various information processing, such as a tablet terminal. The tablet terminal functions as the analysis unit, the storage unit, the input unit, and the output unit described above by executing the programs installed in the tablet terminal.
　図４は、端末２の構成例を示すブロック図である。端末２は、制御部２１、主記憶部２２、通信部２３、表示部２４、入力部２５、及び補助記憶部２６を備える。
　制御部２１は、一又は複数のＣＰＵ（Central Processing Unit）、ＭＰＵ（Micro-Processing Unit）、ＧＰＵ（Graphics Processing Unit）等の演算処理装置を有し、補助記憶部２６に記憶されたプログラムＰを読み出して実行することにより、種々の情報処理、制御処理等を行う。主記憶部２２は、ＳＲＡＭ（Static Random Access Memory）、ＤＲＡＭ（Dynamic Random Access Memory）等の一時記憶領域であり、制御部２１が演算処理を実行するために必要なデータを一時的に記憶する。通信部２３は、通信に関する処理を行うための通信モジュールであり、外部と情報の送受信を行う。表示部２４は、液晶ディスプレイ等の表示画面であり、画像を表示する。入力部２５は、キーボード、マウス等の操作インターフェースであり、作業者から操作入力を受け付ける。補助記憶部２６は、ハードディスク等の不揮発性記憶領域であり、制御部２１が処理を実行するために必要なプログラムＰ（プログラム製品）、その他のデータを記憶している。 FIG. 4 is a block diagram showing a configuration example of the terminal 2. As shown in FIG. The terminal 2 includes a control section 21 , a main storage section 22 , a communication section 23 , a display section 24 , an input section 25 and an auxiliary storage section 26 .
The control unit 21 has an arithmetic processing unit such as one or more CPU (Central Processing Unit), MPU (Micro-Processing Unit), GPU (Graphics Processing Unit), etc., and executes the program P stored in the auxiliary storage unit 26. Various information processing, control processing, etc. are performed by reading and executing the data. The main storage unit 22 is a temporary storage area such as SRAM (Static Random Access Memory) or DRAM (Dynamic Random Access Memory), and temporarily stores data necessary for the control unit 21 to perform arithmetic processing. The communication unit 23 is a communication module for performing processing related to communication, and transmits and receives information to and from the outside. The display unit 24 is a display screen such as a liquid crystal display, and displays images. The input unit 25 is an operation interface such as a keyboard and a mouse, and receives operation inputs from the operator. The auxiliary storage unit 26 is a nonvolatile storage area such as a hard disk, and stores programs P (program products) necessary for the control unit 21 to execute processing and other data.
　なお、端末２は、ＣＤ－ＲＯＭ等の可搬型記憶媒体２ａを読み取る読取部を備え、可搬型記憶媒体２ａからプログラムＰを読み取って実行するようにしても良い。 It should be noted that the terminal 2 may be provided with a reading unit for reading a portable storage medium 2a such as a CD-ROM, and read and execute the program P from the portable storage medium 2a.
（第１実施形態）
　一態様において、本発明におけるシステムは、図５に示されたフロー図に基づいて動作することができる。 (First embodiment)
In one aspect, the system in the present invention can operate according to the flow diagram shown in FIG.
　図５は、本発明のシステムの一態様におけるフロー図を示す。図示されるように、本発明のシステムは、プログラムにより開始されると、（Ｆ１）測定部により細胞の分布を測定し、（Ｆ２）解析部によりシート状細胞培養物の形成度合いを算出し、（Ｆ３）出力部に対して算出値を出力する。そして、（Ｆ４）算出値と設定値とを比較し、（Ｆ５）算出値が設定値を超えていない場合、シート状細胞培養物が未完成である（ｎｏ）と判定して（Ｆ１）に戻る。 FIG. 5 shows a flow diagram in one aspect of the system of the present invention. As illustrated, when the system of the present invention is started by the program, (F1) the measurement unit measures the distribution of cells, (F2) the analysis unit calculates the degree of sheet-like cell culture formation, (F3) Output the calculated value to the output unit. Then, (F4) compare the calculated value and the set value, and (F5) if the calculated value does not exceed the set value, determine that the sheet-like cell culture is incomplete (no) and proceed to (F1) return.
　かかるループ処理により、（Ｆ１）測定、（Ｆ２）解析、（Ｆ３）出力が繰り返えされ、経時的な測定と解析が実行される。算出値が設定値を超えた場合、シート状細胞培養物が完成した（ｙｅｓ）と判定して、（Ｆ６）出力部に対して判定結果を出力して、プログラムを終了する。（Ｆ６）で判定結果を出力した後に、上記の様な剥離部で、シート状細胞培養物の剥離処理を実行することもできる。 Through such loop processing, (F1) measurement, (F2) analysis, and (F3) output are repeated, and chronological measurement and analysis are performed. If the calculated value exceeds the set value, it is determined that the sheet-like cell culture is completed (yes), and (F6) the determination result is output to the output unit, and the program is terminated. After outputting the determination result in (F6), the peeling process of the sheet-like cell culture can be performed at the peeling unit as described above.
　作業者は、（Ｆ３）で出力部に表示された算出値を元に、算出値と設定値との比較（Ｆ４の処理に相当）、シート状細胞培養物の完成、未完成の判定（Ｆ５の処理に相当）を手動で行ってもよい。剥離処理は、上記の様な剥離部でおこなってもよいし、作業者が手動で行ってもよい。（Ｆ３）における算出値は、検量線や学習モデルを使用して予測された、シート状細胞培養物の完成の時刻やカウントダウンであってもよく、その場合、作業者は、前もって完成後に必要な作業の準備を行うことができる。 Based on the calculated value displayed on the output unit in (F3), the operator compares the calculated value with the set value (equivalent to the process of F4), determines whether the sheet-like cell culture is complete or incomplete (F5 (equivalent to the processing of ) may be performed manually. The peeling process may be performed in the peeling section as described above, or may be manually performed by an operator. The calculated value in (F3) may be the time or countdown of the completion of the sheet-like cell culture predicted using a calibration curve or a learning model, in which case the operator is required in advance after completion Be prepared to work.
（第２実施形態）
　一態様において、本発明におけるシステムは、図６に示されたフロー図に基づいて動作することができる。 (Second embodiment)
In one aspect, the system in the present invention can operate according to the flow diagram shown in FIG.
　図６は、本発明のシステムの一態様におけるフロー図を示す。図示されるように、本発明のシステムは、プログラムにより開始されると、（Ｐ１）測定部により細胞の分布を測定し、（Ｐ２）解析部によりシート状細胞培養物の剥離度合いを算出し、（Ｐ３）出力部に対して算出値を出力し、（Ｐ４）算出値と設定値とを比較し、（Ｐ５）算出値が設定値を超えていない場合、シート状細胞培養物の剥離処理が不要である（ｎｏ）と判定して（Ｐ１）に戻る。 FIG. 6 shows a flow diagram in one aspect of the system of the present invention. As illustrated, when the system of the present invention is started by the program, (P1) the measurement unit measures the distribution of cells, (P2) the analysis unit calculates the degree of detachment of the sheet-like cell culture, (P3) Output the calculated value to the output unit, (P4) compare the calculated value with the set value, and (P5) if the calculated value does not exceed the set value, detachment of the sheet-like cell culture is performed. It determines that it is unnecessary (no) and returns to (P1).
　かかるループ処理により、（Ｐ１）測定、（Ｐ２）解析、（Ｐ３）出力が繰り返えされ、経時的な測定が行われる。算出値が設定値を超えた場合、シート状細胞培養物の剥離処理が必要である（ｙｅｓ）と判定して、（Ｐ６）出力部に対して判定結果を出力して、プログラムを終了する。（Ｐ６）で判定結果を出力した後に、シート状細胞培養物を上記の様な剥離部で、剥離処理することもできる。 Through this loop processing, (P1) measurement, (P2) analysis, and (P3) output are repeated to perform measurements over time. If the calculated value exceeds the set value, it is determined that the sheet-like cell culture needs to be detached (yes), (P6) the determination result is output to the output unit, and the program is terminated. After outputting the determination result in (P6), the sheet-like cell culture can be subjected to peeling treatment at the peeling unit as described above.
　（Ｐ４）の設定値は、上記のように、完成状態が１００％であるとき、剥離度合いが１％であるとき、シート化状態が１０１％であるときなど自由に設定することができる。また、（Ｐ２）および（Ｐ３）において、剥離度合いに加えて、形成度合いを算出および出力することもでき、形成度合いが１００％に満たないとき（例えば、９０％）に、剥離が始まったときは、（Ｐ６）において、異常剥離であることや、未完成であること（完成度９０％）を情報として出力してもよい。 The set value of (P4) can be freely set as described above, such as when the completed state is 100%, when the peeling degree is 1%, and when the sheet state is 101%. Further, in (P2) and (P3), in addition to the degree of peeling, the degree of formation can also be calculated and output. In (P6), abnormal peeling or incompleteness (completeness 90%) may be output as information.
　作業者は、（Ｐ３）で出力部に対して出力された算出値を元に、算出値と設定値との比較（Ｐ４の処理に相当）、シート状細胞培養物の剥離処理の判定（Ｐ５の処理に相当）を行ってもよい。剥離処理は、上記の様な剥離部でおこなってもよいし、作業者が手動で行ってもよい。（Ｐ３）における算出値は、経時的な測定から予測された、シート状細胞培養物の剥離処理開始の時刻であってもよく、その場合、作業者は、前もって剥離処理に必要な作業の準備を行うことができる。 Based on the calculated value output to the output unit in (P3), the operator compares the calculated value with the set value (equivalent to the process of P4), determines the detachment process of the sheet-like cell culture (P5 (equivalent to the processing of ) may be performed. The peeling process may be performed in the peeling section as described above, or may be manually performed by an operator. The calculated value in (P3) may be the time to start the detachment treatment of the sheet-like cell culture predicted from the measurement over time, in which case the operator prepares the work necessary for the detachment treatment in advance. It can be performed.
　上記のように、シート状細胞培養物は、周縁部から徐々に剥離が始まるため、測定部による測定は、少なくとも培養容器の底面の周縁部に対して行えばよい。すなわち、測定部は、上記のように、培養容器の側面側から底面方向、および／または底面側から側面方向のように、培養容器の底面の周縁部を斜めに横断するように測定することで、シート状細胞培養物の剥離が、物理的に視野から消失する動きとして検出することもできる。 As described above, the sheet-like cell culture begins to gradually peel off from the peripheral edge, so the measurement by the measurement unit should be performed at least on the peripheral edge of the bottom surface of the culture vessel. That is, as described above, the measurement unit performs measurement so as to obliquely cross the periphery of the bottom surface of the culture vessel, such as from the side surface of the culture vessel toward the bottom surface and/or from the bottom surface side toward the side surface. , the detachment of the sheet-like cell culture can also be detected as a movement that physically disappears from the field of view.
　以上、本発明によれば、細胞の移動や形状変化に関する情報を確実に取得できるため、従来よりも高い精度でシート状細胞培養物のシート化状態を評価することができる。また、本発明によれば、人間による観察等によってでは到底不可能な解析ができるため、従来不可能であったシート化状態の数値化が可能となる。
　本発明のシステムの一態様を説明したが、上記以外の様々な態様が可能であることを理解されたい。したがって、上記の態様を、本発明の思想を逸脱しない範囲で改変した種々の態様もまた本発明の範囲に包含され、かかる改変は当業者にとって理解可能である。
  As described above, according to the present invention, it is possible to reliably acquire information on cell movement and shape change, and therefore it is possible to evaluate the sheet-forming state of a sheet-like cell culture with higher accuracy than in the past. In addition, according to the present invention, since analysis that is absolutely impossible by human observation or the like can be performed, it becomes possible to quantify the state of sheeting, which has been impossible in the past.
Having described one aspect of the system of the present invention, it should be understood that many other aspects are possible. Therefore, various embodiments in which the above embodiments are modified without departing from the spirit of the present invention are also included in the scope of the present invention, and such modifications can be understood by those skilled in the art.

Claims (10)

  1. 　シート状細胞培養物のシート化状態を評価するためのシステムであって、
    　細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得する測定部、および
    　測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出する解析部を含み、
    　測定部は、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するように構成されている、前記システム。     A system for evaluating the sheet state of a sheet-like cell culture,
    including a measurement unit that acquires information about the distribution of cells in the culture vessel in which the cell suspension is seeded, and an analysis unit that analyzes the information acquired by the measurement unit and calculates the degree of detachment of the sheet-like cell culture,
    The above system, wherein the measurement unit is configured to measure cells in the culture vessel from a bottom direction and/or a side direction in contact with the cell suspension in the culture vessel.
  2. 　測定が、培養容器の側面方向から行われる、請求項１に記載のシステム。 The system according to claim 1, wherein the measurement is performed from the lateral direction of the culture vessel.
  3. 　測定が、光検出により行われる、請求項１または２に記載のシステム。 The system according to claim 1 or 2, wherein the measurement is performed by optical detection.
  4. 　測定部は投光器及び受光器であり、
    　投光器は培養容器の底面又は側面に配置され、
    　受光器は培養容器の側面又は底面に配置され、
    　測定が、投光器からシート状細胞培養物の培養面に対して斜めに照射された光を受光器で検出することにより行われる、請求項３に記載のシステム。     The measurement part is a projector and a receiver,
    The projector is arranged on the bottom or side of the culture vessel,
    The light receiver is arranged on the side or bottom of the culture vessel,
    4. The system according to claim 3, wherein the measurement is performed by detecting, with a light receiver, light obliquely emitted from a light projector to the culture surface of the sheet-like cell culture.
  5. 　解析部が、算出値が設定値以上である場合にシート状細胞培養物の剥離処理が必要と判定するように構成されている、請求項１～４のいずれか一項に記載のシステム。 The system according to any one of claims 1 to 4, wherein the analysis unit is configured to determine that detachment processing of the sheet-like cell culture is necessary when the calculated value is equal to or greater than the set value.
  6. 　解析部による解析が、学習済みモデルを用いて行われる、請求項１～５のいずれか一項に記載のシステム。 The system according to any one of claims 1 to 5, wherein the analysis by the analysis unit is performed using a trained model.
  7. 　解析部による解析結果を出力する出力部をさらに含む、請求項１～６のいずれか一項に記載のシステム。 The system according to any one of claims 1 to 6, further comprising an output unit that outputs analysis results by the analysis unit.
  8. 　シート状細胞培養物のシート化状態を評価するためのプログラムであって、
    　細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得する処理、および
    　測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出する処理をプロセッサに実行させ、
    　取得する処理は、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定する処理を含む、前記プログラム。     A program for evaluating the sheet state of a sheet-like cell culture,
    A processor is caused to execute a process of acquiring information about the distribution of cells in the culture vessel in which the cell suspension is seeded, and a process of analyzing the information acquired by the measurement unit and calculating the degree of detachment of the sheet-like cell culture. ,
    The above program, wherein the acquiring process includes measuring the cells in the culture vessel from the bottom direction and/or the side direction in contact with the cell suspension of the culture vessel.
  9. 　シート状細胞培養物のシート化状態を評価するための方法であって、
    　細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得するステップ、および
    　測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出するステップを含み、
    　取得するステップは、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するステップを含む、前記方法。     A method for evaluating the sheet state of a sheet-like cell culture, comprising:
    Obtaining information about the distribution of cells in the culture vessel in which the cell suspension is seeded, and analyzing the information obtained by the measurement unit to calculate the degree of detachment of the sheet-like cell culture,
    The above method, wherein the obtaining step comprises measuring cells in the culture vessel from a bottom direction and/or a side direction in contact with the cell suspension of the culture container.
  10. 　シート状細胞培養物の製造方法であって、
    　細胞懸濁液を播種した培養容器内の細胞の分布に関する情報を取得するステップ、
    　測定部により取得された情報を解析してシート状細胞培養物の剥離度合いを算出するステップ、および
    　算出値が設定値以上である場合にシート状細胞培養物の剥離処理が必要と判定するステップを含み、
    　取得するステップは、培養容器の細胞懸濁液と接触している底面方向および／または側面方向から、培養容器内の細胞を測定するステップを含む、前記製造方法。
          A method for producing a sheet-like cell culture,
    obtaining information about the distribution of cells in the culture vessel seeded with the cell suspension;
    a step of calculating the degree of detachment of the sheet-like cell culture by analyzing information acquired by the measurement unit; including
    The above manufacturing method, wherein the obtaining step includes measuring cells in the culture vessel from a bottom direction and/or a side direction in contact with the cell suspension of the culture vessel.
PCT/JP2023/004074 2022-02-08 2023-02-08 System, program and method for evaluating sheeted state of sheet-shaped cell culture, and method for producing sheet-shaped cell culture WO2023153414A1 (en)

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WO2016152592A1 (en) * 2015-03-25 2016-09-29 テルモ株式会社 Method for evaluating sheet-like cell culture
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Publication number Priority date Publication date Assignee Title
JP2003235540A (en) * 2002-02-20 2003-08-26 Masahito Taya Peeled cell-selecting device, method for selecting peeled cell and its program
WO2016009789A1 (en) * 2014-07-18 2016-01-21 株式会社日立ハイテクノロジーズ Cell culture device and image analysis device
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