WO2006095896A1 - Systeme de surveillance de cellules cultivees - Google Patents

Systeme de surveillance de cellules cultivees Download PDF

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Publication number
WO2006095896A1
WO2006095896A1 PCT/JP2006/304838 JP2006304838W WO2006095896A1 WO 2006095896 A1 WO2006095896 A1 WO 2006095896A1 JP 2006304838 W JP2006304838 W JP 2006304838W WO 2006095896 A1 WO2006095896 A1 WO 2006095896A1
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cells
culture
cell
cultured
gldm
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PCT/JP2006/304838
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English (en)
Japanese (ja)
Inventor
Chiaki Hidai
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Nihon University
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Publication of WO2006095896A1 publication Critical patent/WO2006095896A1/fr

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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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Definitions

  • the present invention relates to a cultured cell monitoring system that can monitor the state of a cultured cell (cell population) over time.
  • the “sarcoma classification method by texture analysis” and the “pattern classification method of pigmented nevus by texture analysis” are already publicly known (Yuriko Murase, Toshiyuki Tanaka, Texture Sarcoma classification by analysis, Proceedings of 1999 IEICE General Conference, No. D-16-6, (2000-03); Toshiyuki Tanaka et al "Pattern Classincation of Nevus with Texture Analysis, The 26th International Conference of the IEEE BMES) These These methods are intended for tumor cells. Then, we focused on the uniformity of the size and shape of the three known moonri 3 ⁇ 4r tables (homogeneous pattern, globular pattern, reticular pattern), and used them as specific parameters.
  • texture analysis which is a processing method, and is used for simple discrimination as a pre-stage to distinguish between benign and malignant tumors.
  • these methods are methods for discrimination based on the photographed cell images, so there is no risk of infection or contamination during the confirmation work as described above, and confirmation can be done in a short time. It is excellent in that it can be done.
  • the problem to be solved by the present invention is that confirmation of the state of culture of target cells over time can be performed easily and in a short time, and there is no risk of infection or contamination of cultured cells at the time of confirmation. It is to provide a culture cell monitoring system.
  • the present inventor has intensively studied to solve the above problems.
  • the texture analysis which is the image processing method described above, is used to monitor the cultured cells over time, and this analysis is performed using the parameters related to the elapsed culture time of the cultured cells as an index.
  • the present invention is as follows.
  • Culture cell monitoring equipped with means for taking images of cultured cells and means for analyzing the texture of the taken cells using the parameters related to the culture time of the cultured cells as an index system.
  • the cultured cells may be human-derived cells, for example, cells for regenerative medicine are preferable.
  • a method for monitoring a cultured cell comprising: a step of taking an image of the cultured cell; and a step of performing a texture analysis on the taken image of the cell using a parameter related to the culture elapsed time of the cultured cell as an index.
  • FIG. 1 is a schematic diagram of the cultured cell monitoring system of the present invention.
  • FIG. 2 is a graph showing a photograph taken in preliminary experiment 1 in Example 1 and an analysis result.
  • FIG. 3 is a view showing a photograph taken in preliminary experiment 2 in Example 1 and an analysis result.
  • FIG. 4 is a graph showing a photograph taken in preliminary experiment 3 in Example 1 and an analysis result.
  • FIG. 5 is a graph showing the results of preliminary experiment 4 in Example 1.
  • FIG. 6 is a graph showing the results of Preliminary Experiment 5 in Example 1.
  • FIG. 7 shows photographs and analysis results taken in preliminary experiment 6 in Example 1.
  • FIG. 8 is a graph showing the analysis results in Monitoring Example 1 in Example 1.
  • FIG. 9 is a photograph showing the results in Example 2 and a graph showing the analysis results.
  • the present invention is characterized in that various parameters in texture analysis, which is an image processing technique, are used for monitoring cultured cells over time.
  • texture analysis which is an image processing technique
  • infection cannot cause contamination.
  • texture analysis includes image analysis methods using a co-occurrence matrix, difference statistics, and density histograms.
  • SGLDM spatial density level-dependent method
  • GLDM density level
  • GLHM concentration histogram method
  • the texture analysis is performed using a parameter related to the culturing time of the cultured cells as an index.
  • a parameter related to the culturing time of the cultured cells as an index.
  • an optimum one can be selected and adopted as appropriate from among the predetermined parameters as the texture feature values described above.
  • the parameters used as the above-mentioned indices can be generally adopted for each type of cultured cell (one type or two or more types). Without being limited thereto, it can be said that the usefulness of the present invention is very high in this respect.
  • the present invention shows extremely high utility especially in the field of regenerative medicine.
  • the field of regenerative medicine several tens to several thousand equivalent to the number of patients It is considered inevitable that there is a need to manage a large number of cultured cell lines at the same time, and there are changes in the number and morphology of cells, the state of proliferation and differentiation, and the presence or absence of defective cells compared to normal cell culture. It is highly necessary to manage many factors such as these simultaneously and with a certain level of reliability. Under these circumstances, the present invention can be said to satisfy these needs all at once, and is extremely useful and exhibits a remarkable effect.
  • the cultured cell monitoring system of the present invention has a means for capturing an image of a cultured cell (hereinafter referred to as “imaging means”) and a means for analyzing a captured cell image (hereinafter referred to as “imaging means”). Analysis system ”), and the texture analysis is performed using a parameter related to the culture elapsed time of the cultured cell as an index.
  • the cultured cells to be monitored are not limited.
  • animal cells, plant cells, insect cells and the like can be used as monitoring targets in the present invention. preferable.
  • animal cells include, but are not limited to, human-derived cells, monkey-derived cells (such as monkey kidney-derived cells (Cos cells (Cosl cells))), mouse-derived cells (mouse vascular endothelial cells (mouse vascular endothelial cells ( Pro5 cells), etc.), and pig-derived cells.
  • monkey-derived cells such as monkey kidney-derived cells (Cos cells (Cosl cells)
  • mouse-derived cells mouse vascular endothelial cells (mouse vascular endothelial cells ( Pro5 cells), etc.
  • pig-derived cells include, but are not limited to, human-derived cells, monkey-derived cells (such as monkey kidney-derived cells (Cos cells (Cosl cells))), mouse-derived cells (mouse vascular endothelial cells (mouse vascular endothelial cells ( Pro5 cells), etc.), and pig-derived cells.
  • the cells derived from humans are not limited, and for example, various tissue cells (oral mucosal cells, adipocytes, chondrocytes, etc.), and tumor cells (benign and malignant cancer cells (derived from human digestive organ cancer) Cells (HCT15 cells) and the like), etc., but cells for regenerative medicine are preferred in that the effect of the present invention can be sufficiently exerted.
  • tissue cells oral mucosal cells, adipocytes, chondrocytes, etc.
  • tumor cells derived from human digestive organ cancer
  • HCT15 cells derived from human digestive organ cancer
  • cells for regenerative medicine can be preferably used for the same reason.
  • Examples of cells for regenerative medicine include various pluripotent stem cells (specifically, all Preferred stem cells and tissue stem cells), but not limited thereto.
  • Totipotent stem cells are cells that can differentiate into all somatic cells, and examples include, but are not limited to, ES cells (embryonic stem cells / embryonic stem cells) and EG cells.
  • a tissue stem cell is a cell that can differentiate into a plurality of cell types constituting a specific organ or tissue, and examples include neural stem cells, hematopoietic stem cells (bone marrow cells, etc.), and fat cells. There is no limitation.
  • the cultured cells to be monitored are not limited as long as they are cultured in a well-known culture container as appropriate according to the type of the cultured cells. Is preferably transparent (preferably colorless and transparent). Examples of the culture container include a plastic petri dish, a plastic flask, a glass petri dish, and the like. In addition, the culture vessel containing cells is generally placed in a known incubator (incubator) that is temperature-controlled and cultured.
  • the means for taking an image of the cultured cell is not limited, but at least a device that can magnify the cultured cell (device (a)) and a device that can take a still image of the cultured cell (device (b) ), And equipment (equipment (c)) that can construct image data (electronic data) based on a photographed still image that can be used in the analysis means described later.
  • These devices (devices (a) to (c)) may be devices in which all or any of the two forces are integrated (devices having substantially the functions of each device), or at least The two may be separate devices and is not limited.
  • Examples of the device (a) include, but are not limited to, various microscopes such as an inverted microscope, an optical microscope, and a laser microscope.
  • the device (b) is not limited, and examples thereof include various cameras.
  • the device (C) is not limited, and examples thereof include a scanner.
  • the device having the functions of the devices (b) and (C) is not limited, and examples thereof include a digital camera (including digital video with a camera function).
  • the photographing means may further include other devices such as an autofocus device and an automatic exposure time measurement device.
  • the photographing means may be arranged inside or outside the incubator (incubator), and is not limited.
  • a plurality of imaging means (part or all of them, in particular, the device (a)) may be provided in combination with the number of culture vessels (number of samples), or a plurality of imaging means may be moved to The culture vessel may be monitored and is not limited.
  • the imaging interval of the cultured cells by the imaging means may be set as appropriate depending on the type of the cultured cells to be monitored, and is not limited.
  • the interval is 1 day from the first day of the culture start. It may be 1 hour interval or 1 minute interval.
  • the means for texture analysis of the photographed cultured cell image is not limited, and examples thereof include a computer that can perform texture analysis on the image data obtained by the photographing means.
  • Existing software can be used for texture analysis, for example, Poplmaging (Digital 'Being. kids').
  • the computer may also control other means such as, for example, control of photographing conditions and position of an electronic device (photographing means) such as a digital camera.
  • photographing means such as a digital camera.
  • Existing software can be used to control the digital camera. Examples include Aquacosmos (manufactured by Hamamatsu Photonics), NIH Image (manufactured by NIH), and Scion Image (manufactured by Scion Corporation).
  • Aquacosmos manufactured by Hamamatsu Photonics
  • NIH Image manufactured by NIH
  • Scion Image manufactured by Scion Corporation
  • texture analysis spatial density level-dependent method, density level difference method, and density histogram method.
  • a co-occurrence matrix with elements 2, ',', and n-1) is obtained, and the texture features are obtained from the matrix.
  • texture parameters are calculated for five parameters: “energy, entropy, correlation, local uniformity, and inertia”.
  • the density histogram method is a method for obtaining texture features from a density histogram P (i) normalized so that the whole becomes 1.
  • the texture features are calculated for the four parameters “average, variance, skewness, and kurtosis”.
  • the skewness is a parameter indicating the symmetry shape characteristic of the density histogram, that is, how much the density histogram has deviated from the symmetrical shape.
  • the kurtosis is the distribution shape characteristic of the density histogram, In other words, it is a parameter that represents how much the concentration histogram distribution is concentrated around the average value.
  • a calculation method (calculation formula) of each parameter a known method is adopted.
  • the image data obtained by the photographing means is generally subdivided into equal areas (divided into squares), and the parameter values as described above are measured for each square. , Not limited to this, The entire image data may be measured.
  • parameters related to the elapsed time of cultured cells are effective for monitoring cultured cells over time as an index.
  • parameters related to the elapsed time of cultured cells It is important to be carried out using as an index.
  • “to be performed using parameters related to the elapsed time of cultured cells as an index” generally means that measurement of parameters in texture analysis is performed only for parameters that can be used as the above indexes from the beginning (this However, the present invention is not limited to this. For example, various parameters can be measured over time, and the parameters that can be used as the index from the measurement results. Is also included to select appropriately.
  • the parameter related to the culture elapsed time of the cultured cell is substantially equal to the parameter value measured over time (Y axis) plotted against the culture elapsed time (X axis).
  • the measured value (Y axis) is substantially constant with respect to the culture elapsed time (X axis).
  • a parameter showing an increasing or decreasing tendency (proportional relationship) at a ratio of is not constant, or a parameter in which no substantial change is observed Etc.
  • the parameter is unique depending on the type of cultured cells, and is not limited to a specific type. Further, it may be one type or two or more types and is not limited. (4) Other means
  • the cultured cell monitoring system of the present invention may include other means in addition to the imaging means and analysis means, and is not limited.
  • These may be provided alone or in combination of two or more, and are not limited.
  • the means for notifying the abnormality is not limited, but examples thereof include a device such as an alarm device. Specifically, it is preferable to operate by sending a signal from a computer used for the analyzing means.
  • FIG. 1 For the arrangement of each means in the cultured cell monitoring system of the present invention, the schematic diagram shown in FIG. 1 can be referred to.
  • the usage mode of the cultured cell monitoring system of the present invention is not limited, but for example, the following modes ⁇ and ( ⁇ ) are preferable.
  • the number of samples of cells to be monitored needs to be at least 3 or more, and is preferably 10 or more, more preferably, in order to improve reliability. 20 or more, particularly preferably 100 or more.
  • Culture of cells to be monitored (three or more samples) is started under conditions that allow the desired culture progress, and various parameters in each sample are measured over time using the system of the present invention.
  • the behavior of the measured value with respect to the culture elapsed time is more than half (for example, 1/2 or more, preferably 2/3 or more, more preferably 19/20 or more More preferably 99/100 or more), a common parameter is recognized as one of the above-mentioned “parameters related to the culturing time of cultured cells”, Two or more parameters can be selected. After selection, measure various parameters and create a graph in the same way. (You may limit to the selected parameters.) For at least one of the selected parameters, the other samples in the majority sample above Samples that show a value significantly different from this graph can be judged to have deviated from the normal culture state (the desired culture progress state was not obtained).
  • the optimal imaging conditions differ depending on the type of cultured cells, but automatic analysis of many types of cells becomes difficult if the analysis results vary greatly depending on the imaging conditions. Therefore, when using the cultured cell monitoring system of the present invention, it is preferable to select in advance parameters that are not influenced by the imaging conditions or have little influence.
  • the imaging conditions that should be considered are not limited, but examples include exposure time and focus.
  • the cultured cell monitoring method of the present invention includes a step of capturing an image of a cultured cell (hereinafter referred to as “imaging step”) and a step of analyzing a texture of the captured cell image (hereinafter referred to as “analysis step”). And the texture analysis is performed using a parameter related to the culture elapsed time of the cultured cells as an index.
  • the cultured cells to be monitored in the cultured cell monitoring method of the present invention are not limited, but the same explanation as the system of the present invention can be preferably applied.
  • the process of taking an image of the cultured cell is not limited, but at least a device capable of magnifying the cultured cell (device (a)), a device capable of photographing a still image of the cultured cell (device (b)), and This can be done using equipment (equipment (c)) that can construct image data (electronic data) based on the captured still image that can be used in the analysis means described later.
  • equipment equipment
  • These devices (devices (a) to (c)) may be devices in which all or any two of them are integrated (devices having substantially the functions of each device), or at least Both can be devices that are separate and are not limited.
  • the imaging interval of the cultured cells The same explanation as the system can be preferably applied.
  • texture analysis can be performed on the image data obtained by the imaging process (with software for texture analysis). Can be carried out using the data.
  • texture analysis there are three methods for texture analysis (spatial density level dependent method, density level difference method, and density histogram method).
  • the same description as the system of the present invention can be preferably applied to the details of these methods and the explanation regarding the segmentation of image data.
  • “texture analysis is performed using the parameters effective for monitoring the cultured cells over time (specifically, parameters related to the elapsed culture time of the cultured cells) as an index, which is a feature of the method of the present invention.
  • the description similar to that of the system of the present invention can also be preferably applied to the explanation regarding the parameters.
  • the cultured cell monitoring method of the present invention may include other steps in addition to the imaging step and the analysis step, and is not limited.
  • the usage mode of the cultured cell monitoring method of the present invention is not limited, but the same examples and explanations as those of the system of the present invention can be preferably applied. (6) Influence of shooting conditions
  • a cultured cell monitoring system as shown in the schematic diagram of Fig. 1 was prepared.
  • an imaging method an inverted microscope (manufactured by Olympus, product name: IX 71), and a digital camera (manufactured by Hamamatsu Photonitas, product name: ORCA-) connected so as to be able to photograph images with the microscope are used.
  • the analysis method is a computer that can analyze the image data (Epson, product name: Endeavor), and image analysis software (Digital Bi-Fung kids, product name, : Poplmaging), and the above imaging means can be placed in an incubator where the temperature can be set and adjusted as required.
  • culture and texture analysis were conducted using the above monitoring system, although not particularly mentioned.
  • Pro5 cells were intentionally changed to a cultured state (abnormality: trouble) by removing serum from the culture medium during the culture, and the changes were monitored over time by texture analysis and evaluated.
  • Pro5 cells were seeded, and photographs were taken every day from the next day, and SGLDM entropy, SGLDM correlation, SGLDM local uniformity, GLDM contrast, GLDM angular secondary moment, GLDM entropy, and GLDM average were evaluated.
  • the results (graph) are shown in Fig. 5A.
  • the parameters related to the elapsed time of cultured cells include SGLDM entropy, SGLDM local uniformity, GLDM contrast, GLDM second moment, and GLDM. You can select the pea pea and the GLDM average.
  • the parameters related to the elapsed time of the cultured cells are: SGLDM inlet mouthpiece, SGLDM local uniformity, GLDM contrast, GLDM second moment by angle , GLDM entry, and GLDM average can be selected.
  • HCT15 cells were seeded, and photographs were taken every day from the next day, and SGLDM entropy, SGLDM correlation, SGLDM local uniformity, GLDM contrast, GLDM angular second moment, GLDM entropy, and GLDM average were evaluated.
  • the results (graph) are shown in Fig. 5C.
  • the parameters related to the elapsed time of cultured cells include SGLDM entropy, SGLDM local uniformity, GLDM contrast, and GLDM second moment by angle.
  • SGLDM entropy SGLDM local uniformity
  • GLDM contrast GLDM contrast
  • GLDM second moment by angle GLDM Mouth pea
  • GLDM average can be selected
  • the 16 numbers corresponding to each area were color-coded by larger and smaller numbers using the numerical value that can be divided into 8 as the boundary value (Figure 7B). As shown in the photograph in Fig. 7A, the upper right part is occupied by Pro5 cell colonies and the lower left part is occupied by COS cell colonies. At the boundary, an area where both cells coexist diagonally from the upper left to the lower right.
  • the results of texture analysis were divided into the following three patterns.
  • ⁇ Monitoring example 1 Monitoring the culture state of Pro5 cells (Detection of incubator failure using GLDM contrast)>
  • Pro5 cells were seeded in a culture dish, and observation was started after 1 day. Since it is known from the analysis of the normal group that the contrast of the GLDM method is suitable for the evaluation of cell proliferation as described above (see Preliminary Experiment 4 (1)), the culture state is monitored using the contrast as an index. Went. As a sample, after 2 days of culturing, the incubator was turned off to create a situation where the cell culture state was intentionally abnormal.
  • Cosl cells were seeded in 6 culture dishes, and the culture was started. After 2 days of observation, 10ng / ml epidermal growth factor was added to 3 cultures (non-control group). The changes were compared with the remaining 3 sheets (control group) to which no epidermal growth factor was added. A photograph is taken and observed every day after the addition, and based on the photograph (image data), “GLDM contrast, GLDM second moment, The culture state was monitored by texture analysis using GLDM entropy, GLDM average, SGLDM entropy, and SGLDM local uniformity as indices.
  • FIGS. 9A and 9B The photographs on the second day after the addition (that is, the fourth day of culture) are shown in FIGS. 9A and 9B.
  • Fig. 9A shows cells in the control group
  • Fig. 9B shows cells to which epidermal growth factor has been added (non-control group cells).
  • the cells in the non-control group clearly changed in cell shape 2 days after addition, indicating that the widened fibroblast-like shape has increased in cell thickness and differentiated into epithelial-like cells.
  • the culture state of the target cell over time can be confirmed easily and in a short time, and there is no risk of infection or contamination of the cultured cell at the time of confirmation. It is possible to provide a monitoring system.

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Abstract

L’invention concerne la fourniture d’un système de surveillance de cellules cultivées qui comprend un moyen pour photographier une image des cellules cultivées, et un moyen pour analyser la texture de l’image des cellules cultivées ainsi photographiées en utilisant un paramètre associé au temps de culture des cellules cultivées.
PCT/JP2006/304838 2005-03-08 2006-03-07 Systeme de surveillance de cellules cultivees WO2006095896A1 (fr)

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WO2012115153A1 (fr) * 2011-02-25 2012-08-30 株式会社ニコン Procédé d'évaluation de cellules, méthode de culture de cellules, dispositif d'évaluation de cellules, incubateur, programme d'évaluation de cellules, programme de classification de colonies, procédé de culture de cellules souches, dispositif d'évaluation de cellules souches et programme d'évaluation de cellules souches
WO2018055762A1 (fr) * 2016-09-26 2018-03-29 オリンパス株式会社 Système de mesure de l'état de cellules
JP2018108064A (ja) * 2017-01-06 2018-07-12 オリンパス株式会社 細胞観察システム
JP2019000026A (ja) * 2017-06-14 2019-01-10 オリンパス株式会社 細胞培養モニタリングシステム
CN110099995A (zh) * 2017-01-06 2019-08-06 奥林巴斯株式会社 细胞观察系统
WO2020054195A1 (fr) * 2018-09-12 2020-03-19 Jsr株式会社 Système d'imagerie analytique pour organoïdes, procédé et programme correspondants
JP2020094925A (ja) * 2018-12-13 2020-06-18 住友電気工業株式会社 品質評価方法

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WO2012115153A1 (fr) * 2011-02-25 2012-08-30 株式会社ニコン Procédé d'évaluation de cellules, méthode de culture de cellules, dispositif d'évaluation de cellules, incubateur, programme d'évaluation de cellules, programme de classification de colonies, procédé de culture de cellules souches, dispositif d'évaluation de cellules souches et programme d'évaluation de cellules souches
WO2018055762A1 (fr) * 2016-09-26 2018-03-29 オリンパス株式会社 Système de mesure de l'état de cellules
JP2021151257A (ja) * 2017-01-06 2021-09-30 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
JP2018108064A (ja) * 2017-01-06 2018-07-12 オリンパス株式会社 細胞観察システム
CN110099995A (zh) * 2017-01-06 2019-08-06 奥林巴斯株式会社 细胞观察系统
EP3567096A4 (fr) * 2017-01-06 2020-10-21 Olympus Corporation Système d'observation de cellules
US11037293B2 (en) 2017-01-06 2021-06-15 Olympus Corporation Cell observation system
JP7196238B2 (ja) 2017-01-06 2022-12-26 株式会社エビデント 細胞観察システムおよび細胞観察方法
JP2019000026A (ja) * 2017-06-14 2019-01-10 オリンパス株式会社 細胞培養モニタリングシステム
WO2020054195A1 (fr) * 2018-09-12 2020-03-19 Jsr株式会社 Système d'imagerie analytique pour organoïdes, procédé et programme correspondants
JP2020094925A (ja) * 2018-12-13 2020-06-18 住友電気工業株式会社 品質評価方法
CN113167719A (zh) * 2018-12-13 2021-07-23 住友电气工业株式会社 质量评估方法
CN113167719B (zh) * 2018-12-13 2024-06-18 住友电气工业株式会社 质量评估方法

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