WO2023153414A1 - Système, programme et procédé pour évaluer l'état d'exfoliation d'une culture cellulaire en forme de feuille, et procédé pour produire une culture cellulaire en forme de feuille - Google Patents

Système, programme et procédé pour évaluer l'état d'exfoliation d'une culture cellulaire en forme de feuille, et procédé pour produire une culture cellulaire en forme de feuille Download PDF

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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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English (en)
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

La présente invention concerne un système pour évaluer l'état d'exfoliation d'une culture cellulaire en forme de feuille, ledit système comprenant : une unité de mesure pour obtenir des informations sur la distribution des cellules dans un récipient de culture dans lequel une suspension cellulaire est ensemencée ; et une unité d'analyse pour analyser les informations obtenues par l'unité de mesure et calculer le degré de décollement de la culture cellulaire en forme de feuille. L'unité de mesure est conçue pour mesurer les cellules dans le récipient de culture dans les sens inférieur et/ou latéral du récipient de culture en contact avec la suspension cellulaire.
PCT/JP2023/004074 2022-02-08 2023-02-08 Système, programme et procédé pour évaluer l'état d'exfoliation d'une culture cellulaire en forme de feuille, et procédé pour produire une culture cellulaire en forme de feuille WO2023153414A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003235540A (ja) * 2002-02-20 2003-08-26 Masahito Taya 剥離細胞選別装置、剥離細胞選別方法及びそのプログラム
WO2016009789A1 (fr) * 2014-07-18 2016-01-21 株式会社日立ハイテクノロジーズ Dispositif de culture cellulaire et dispositif d'analyse d'images
WO2016152592A1 (fr) * 2015-03-25 2016-09-29 テルモ株式会社 Procédé d'évaluation de culture de cellules en forme de feuille
JP2020156419A (ja) * 2019-03-27 2020-10-01 オリンパス株式会社 細胞観察システムおよび細胞観察方法
WO2021261140A1 (fr) * 2020-06-22 2021-12-30 株式会社片岡製作所 Dispositif de traitement de cellules, dispositif d'apprentissage et dispositif de proposition de modèle appris

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003235540A (ja) * 2002-02-20 2003-08-26 Masahito Taya 剥離細胞選別装置、剥離細胞選別方法及びそのプログラム
WO2016009789A1 (fr) * 2014-07-18 2016-01-21 株式会社日立ハイテクノロジーズ Dispositif de culture cellulaire et dispositif d'analyse d'images
WO2016152592A1 (fr) * 2015-03-25 2016-09-29 テルモ株式会社 Procédé d'évaluation de culture de cellules en forme de feuille
JP2020156419A (ja) * 2019-03-27 2020-10-01 オリンパス株式会社 細胞観察システムおよび細胞観察方法
WO2021261140A1 (fr) * 2020-06-22 2021-12-30 株式会社片岡製作所 Dispositif de traitement de cellules, dispositif d'apprentissage et dispositif de proposition de modèle appris

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