WO2015141059A1 - 薬効評価方法および薬効評価のための画像処理装置 - Google Patents
薬効評価方法および薬効評価のための画像処理装置 Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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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
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T11/00—2D [Two Dimensional] image generation
- G06T11/003—Reconstruction from projections, e.g. tomography
- G06T11/006—Inverse problem, transformation from projection-space into object-space, e.g. transform methods, back-projection, algebraic methods
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/60—Analysis of geometric attributes
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/60—Analysis of geometric attributes
- G06T7/62—Analysis of geometric attributes of area, perimeter, diameter or volume
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N13/00—Stereoscopic video systems; Multi-view video systems; Details thereof
- H04N13/20—Image signal generators
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10072—Tomographic images
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10072—Tomographic images
- G06T2207/10101—Optical tomography; Optical coherence tomography [OCT]
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/30—Subject of image; Context of image processing
- G06T2207/30004—Biomedical image processing
- G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N13/00—Stereoscopic video systems; Multi-view video systems; Details thereof
- H04N13/10—Processing, recording or transmission of stereoscopic or multi-view image signals
- H04N13/106—Processing image signals
- H04N13/111—Transformation of image signals corresponding to virtual viewpoints, e.g. spatial image interpolation
Definitions
- the present invention relates to a medicinal effect evaluation method for evaluating the medicinal effect of a chemical substance on cells cultured in a medium and an image processing apparatus suitable for the medicinal effect evaluation.
- screening is performed to find drugs that have a medicinal effect on specific cells, such as cancer cells.
- An example of such a screening technique is described in Japanese Patent Application Laid-Open No. 2011-062166.
- a chemical substance that is a drug candidate is administered to cultured cells, and cell changes are observed.
- a reagent that exhibits a specific biochemical reaction depending on cell activity is added together with a candidate drug. And the activity of a cell is determined by measuring the quantity of the substance and luminescence produced
- an ATP assay and an MTT assay are known.
- a technique for imaging cells using a microscope or the like has also been proposed.
- the focal position of the microscopic optical system is changed in multiple steps in the depth direction, and imaging is performed each time. . Thereby, a pseudo three-dimensional image of the cell is obtained.
- This technique is suitable for imaging a cell cultured (planar culture) in a state of adhering to the bottom surface of the container into which the culture solution has been injected.
- the present invention has been made in view of the above problems, and an object of the present invention is to provide a technique that makes it possible to more accurately evaluate what kind of medicinal effect a chemical substance exerts on a cell clump.
- One aspect of the present invention is a medicinal effect evaluation method for evaluating a medicinal effect of a chemical substance on a cell clump, wherein the cell clump held in a liquid carried in a container has a cross-section that substantially coincides with a vertical plane.
- the medicinal effect of the chemical substance is evaluated based on the feature amount of the cell agglomeration obtained from the tomographic image in the vertical cross section of the cell agglomeration in the substantially vertical cross section.
- a cell cluster composed of highly active cells exhibits a shape with a relatively high sphericity in a liquid such as a culture solution.
- a liquid such as a culture solution.
- changes such as contraction of the cell clumps or decrease in the sphericity thereof appear.
- such a shape change accompanying the collapse of the cell clump is likely to occur in the lower part or the lower part of the cell clump. That is, the cell clump that cannot maintain the connection between cells collapses downward due to gravity, and the dead cell is released from the clump and falls.
- the feature amount of the cell agglomeration is obtained from a tomographic image obtained by tomographic imaging at a cross section substantially coincident with the vertical plane, and the medicinal effect of the chemical substance is evaluated based on the result.
- an image acquisition means for acquiring a plurality of tomographic images obtained by tomographic imaging of a cell conglomerate in a liquid in a cross section substantially coincident with a vertical plane; 3D image creation means for creating a 3D image of the surface of the cell clump based on the tomographic image; and feature quantity calculation means for calculating the feature quantity of the cell clump based on the plurality of tomographic images or the 3D image.
- the size of the cell agglomeration is measured based on a two-dimensional image (for example, an image obtained by imaging the cell agglomeration from above), and growth or weakness has been observed. The presence or absence was judged.
- the image processing apparatus of the present invention has a function of creating a three-dimensional image of a cell clump from a plurality of tomographic images having a cross section in a substantially vertical direction, and further calculating a feature amount of the cell clump. Therefore, it is extremely effective for carrying out the above-described drug efficacy evaluation method.
- the image processing apparatus of the present invention can provide accurate information to a user who wants to evaluate the efficacy of a chemical substance, and can support the work very effectively.
- FIG. 1 is a diagram showing an embodiment of an image processing apparatus according to the present invention.
- the image processing apparatus 1 can provide useful information for performing the medicinal effect evaluation method according to the present invention. With this function, the image processing apparatus 1 can extremely effectively support the implementation of the medicinal effect evaluation method by the user.
- the configuration of the image processing apparatus 1 and an embodiment of the medicinal effect evaluation method of the present invention that can be performed using the apparatus will be described in order.
- XYZ orthogonal coordinate axes are set as shown in FIG.
- the XY plane represents a horizontal plane
- the Z axis represents a vertical axis. More specifically, the (+ Z) direction represents a vertically upward direction.
- the image processing apparatus 1 performs tomographic imaging of spheroids (cell clumps) cultured in a liquid (for example, a culture solution). And the image processing apparatus 1 image-processes the tomographic image obtained by this, and produces the three-dimensional image of a spheroid. Further, the image processing apparatus 1 calculates a feature amount that quantitatively indicates the appearance feature of the spheroid based on the tomographic image or the stereoscopic image.
- the image processing apparatus 1 has a well plate (also referred to as a microplate) WP in which a large number of recesses (wells) W capable of supporting a liquid are formed on the upper surface of a plate-like member, with the opening surface of the well W facing upward.
- a holding unit 10 that holds the substantially horizontal posture is provided.
- a predetermined amount of an appropriate culture solution is injected in advance into each well W of the well plate WP, and the spheroid Sp is cultured on the bottom surface Wb of the well W in the solution.
- spheroids Sp are described only in some wells W, but spheroids Sp are cultured in each well W.
- the imaging unit 20 is disposed above the well plate WP held by the holding unit 10.
- the imaging unit 20 can capture a tomographic image of an imaging object in a non-contact and non-destructive (non-invasive) manner.
- an optical coherence tomography (OCT) apparatus is used.
- OCT optical coherence tomography
- an imaging unit 20 that is an OCT apparatus includes a light source 21 that generates illumination light for an imaging target, a beam splitter 22 that splits light from the light source 21, an objective lens 23, and a reference mirror 24. And a photodetector 25 and a casing 26 that holds and accommodates these together.
- the image processing apparatus 1 further includes a control unit 30 that controls the operation of the apparatus and a scanning drive mechanism 40 that drives the movable portion of the imaging unit 20.
- the control unit 30 includes a central processing unit (CPU) 31, an A / D converter 32, a 3D restoration unit 33, a feature amount calculation unit 34, an interface (IF) unit 35, an image memory 36, and a memory 37.
- the CPU 31 controls the operation of the entire apparatus by executing a predetermined control program, and the control program executed by the CPU 31 and data generated during the processing are stored in the memory 37.
- the A / D converter 32 converts a signal output from the photodetector 25 of the imaging unit 20 according to the amount of received light into digital image data.
- the 3D restoration unit 33 creates a stereoscopic image (3D image) of the imaged cell cluster based on the image data of a plurality of tomographic images captured by the imaging unit 20.
- the feature amount calculation unit 34 quantitatively determines the morphological features of the cell cluster based on one or more tomographic images captured by the imaging unit 20 or the image data of the stereoscopic image created by the 3D restoration unit 33. The feature quantity shown is calculated.
- the image data of the tomographic image captured by the imaging unit 20 and the image data of the stereoscopic image created by the 3D restoration unit 33 are stored and saved in the image memory 36.
- the interface unit 35 is responsible for communication between the image processing apparatus 1 and the outside. Specifically, the interface unit 35 has a communication function for communicating with an external device, and a user interface function for receiving an operation input from the user and notifying the user of various information. For this purpose, an input device 351 and a display unit 352 are connected to the interface unit 35.
- the input device 351 is, for example, a keyboard, a mouse, a touch panel, or the like that can accept operation inputs related to device function selection, operation condition setting, and the like.
- the display unit 352 displays various processing results such as a tomographic image captured by the imaging unit 20, a stereoscopic image created by the 3D restoration unit 33, and a feature value calculated by the feature value calculation unit 34, for example, a liquid crystal display Is provided.
- the scanning drive mechanism 40 causes the imaging unit 20 to perform a predetermined scanning movement in response to a control command given from the CPU 31.
- a tomographic image of a cell cluster that is an imaging target is acquired by a combination of the scanning movement of the imaging unit 20 executed by the scanning drive mechanism 40 and the detection of the amount of received light by the photodetector 25. Is done.
- FIG. 2A and 2B are diagrams for explaining the imaging principle of the image processing apparatus. More specifically, FIG. 2A is a diagram showing an optical path in the imaging unit 20, and FIG. 2B is a diagram schematically showing a state of tomographic imaging of a spheroid.
- FIG. 2A the description of the casing 26 and the objective lens 23 that is equivalent to a general objective lens in the imaging optical system among the components of the imaging unit 20 is omitted. Yes.
- the imaging unit 20 functions as an optical coherence tomography (OCT) apparatus.
- OCT optical coherence tomography
- a low coherence light beam L1 is emitted from a light source 21 having a light emitting element such as a laser diode or a light emitting diode.
- the light beam L 1 enters the beam splitter 22, a part of the light L 2 indicated by the broken line arrow is directed to the well W, and a part of the light L 3 indicated by the one-dot chain line arrow is directed to the reference mirror 24.
- the light L2 directed toward the well W is incident on the spheroid Sp in the culture solution carried by the well W and is reflected by the spheroid Sp. If the spheroid Sp is not transparent to the light beam L2, the light beam L2 is reflected on the surface of the spheroid Sp. On the other hand, when the spheroid Sp has a certain degree of transparency with respect to the light beam L2, the light beam L2 enters the spheroid Sp and is reflected by the internal structure. By using, for example, near infrared rays as the light beam L2, it is possible to make incident light reach the inside of the spheroid Sp.
- the optical path length is a depth corresponding to the optical path length of the reflected light from the reference mirror 24 ( Only the reflected light from the reflecting surface (position in the Z direction) interferes with the reflected light from the reference mirror 24.
- the photodetector 25 detects the interference light, it is possible to selectively detect the reflected light from the reflecting surface having a specific depth corresponding to the position of the reference mirror 24 in the spheroid Sp.
- reflected light from an arbitrary depth of the spheroid Sp can be detected. This is combined with scanning in the X direction of the light L2 incident on the well W, and interference light is detected by the photodetector 25 as needed.
- a tomographic image of the spheroid Sp whose cross section is a vertical plane parallel to the XZ plane can be captured.
- the relative position of the imaging unit 20 with respect to the well W is changed in multiple stages in the Y direction, and a tomographic image is captured each time.
- a large number of tomographic images It obtained by tomographic imaging of the spheroid Sp in a cross section parallel to the XZ plane can be obtained.
- image data with sufficient resolution to grasp the three-dimensional structure of the spheroid Sp can be obtained.
- the scanning movement of each part in the imaging unit 20 is realized by the operation of the scanning drive mechanism 40 that receives a control command from the CPU 31.
- FIG. 3 is a flowchart showing the operation of the image processing apparatus.
- the well plate WP carrying the spheroid Sp to be imaged together with the culture solution is set on the holding unit 10 by the user or the transport robot (step S101).
- the CPU 31 controls the imaging unit 20 and the scanning drive mechanism 40 to perform tomographic imaging of the spheroid Sp in the well W (step S102).
- the incident position of the light beam on the spheroid Sp changes in the X direction by scanning the light beam. Further, the position of the reference mirror 24 is changed, so that the position in the Z direction of the light receiving surface that receives the reflected light is changed.
- a tomographic image of the spheroid Sp whose cross section is a plane parallel to the XZ plane, that is, a vertical plane perpendicular to the Y direction is obtained.
- the tomographic image of the spheroid Sp in each cross section is taken while the Y direction position of the cross section is changed by the imaging unit 20 moving in the Y direction with respect to the well W. By repeating this, a large number of tomographic images are acquired for cross sections whose positions are different from each other in the Y direction.
- the 3D restoration unit 33 creates 3D image data corresponding to the three-dimensional image of the spheroid Sp (step S103).
- 3D image data can be obtained by interpolating tomographic image data discretely acquired in the Y direction in the Y direction. Since a technique for creating 3D image data from tomographic image data has already been put into practical use, detailed description thereof will be omitted.
- FIGS. 4A and 4B are diagrams showing examples of tomographic images and stereoscopic images.
- a three-dimensional image showing the overall appearance of the spheroid Sp from a large number of tomographic images (two-dimensional image) I2 (FIG. 4A) obtained by imaging the spheroid Sp in a cross section parallel to the XZ plane while changing the position in the Y direction.
- I3 (FIG. 4B) is created.
- the surface of the spheroid Sp that is, the interface between the inside of the spheroid Sp and the culture solution clearly appears.
- the arc-shaped white streaks appearing at the bottom of the image is an image of the bottom surface Wb of the well W.
- the bottom surface Wb of the well W is slightly depressed toward the center, it appears as such an arc-shaped image.
- the white flat image at the bottom of the image in FIG. 4B is slightly depressed toward the center. The same applies to the subsequent drawings.
- the 3D image data created from the tomographic image in this way is obtained by associating the coordinates of each pixel in the virtual XYZ pixel space with the pixel value.
- 3D image data can be used for various processes thereafter. For example, an image corresponding to an image obtained by viewing the spheroid Sp from various viewing directions is created by image processing and displayed on the display unit 352. By doing so, the user can observe the outer shape and the surface shape as if they were looking from an arbitrary direction with the spheroid in front of them.
- the surface of the spheroid Sp has fine irregularities corresponding to the cell interface.
- a determination is made based on the overall shape characteristics of the spheroid Sp. Therefore, such fine unevenness can be an error factor in quantitatively expressing the characteristics of the spheroid Sp. Therefore, an approximate curved surface that approximates the surface of the spheroid Sp to a simpler surface, that is, a curved surface with less unevenness is obtained (step S104).
- Various approximate calculations can be considered as the calculation method, and an example thereof will be described later.
- the obtained approximate curved surface is a curved surface representing the envelope outer shape of the spheroid Sp. Although this approximate curved surface has little information on the state of individual cells constituting the spheroid Sp, the shape characteristics of the entire spheroid Sp can be clearly shown.
- the feature amount calculation unit 34 calculates a feature amount that quantitatively indicates the feature of the spheroid Sp (step S105).
- the medicinal efficacy of the chemical substance is evaluated depending on how the shape of the spheroid Sp to which the chemical substance that is a drug candidate is administered changes.
- normal spheroids have a nearly spherical shape in the culture medium, while spheroids damaged by chemical substances contract or lose shape. Therefore, a feature quantity that can quantitatively detect such a change in outer shape is used.
- the diameter, volume, and surface area of the spheroid, the curvature and radius of curvature of the spheroid, the sphericity of the spheroid, and the like are calculated as the feature quantities.
- a least square approximation is performed to obtain a smooth curved surface from the 3D image data.
- the transpose matrix is used to convert the equation into an equation having a 6 ⁇ 6 normal matrix, and the unknowns a to f may be obtained.
- the curvature of the curved surface is expressed by using two of the Gaussian curvature K and the planar curvature H.
- the Gaussian curvature K and the plane curvature H can be defined by the following equations.
- the curvature in a pixel space in which coordinates are discretely expressed in units of pixels it is possible to perform numerical calculation by replacing the differentiation in (Equation 9) with a difference in pixel pitch.
- FIG. 5 is a flowchart showing a drug efficacy evaluation method in this embodiment.
- an appropriate culture solution is injected into each well W of the well plate WP, and target cells are cultured therein to produce a spheroid Sp (step S201).
- a predetermined amount of each chemical substance to be evaluated is administered to each well W (step S202).
- the image processing apparatus 1 converts the spheroid Sp to which the chemical substance has been administered into image data (step S203). That is, the image processing apparatus 1 executes tomographic image capturing and calculation based on image data obtained thereby. Imaging may be performed only once after a predetermined time has elapsed since the chemical substance was administered. Also, so-called time-lapse imaging may be performed in which imaging is performed a plurality of times at regular time intervals. When the tomographic image data, stereoscopic image data, and feature amount of the spheroid Sp are obtained by the image processing apparatus 1, the medicinal effect of the chemical substance is comprehensively evaluated based on these information (step S204).
- the volume of the spheroid Sp can be calculated by integrating the cross-sectional area of the spheroid Sp in a certain cross-sectional direction in the direction perpendicular to the cross-sectional direction. For example, it is also possible to directly calculate from a plurality of tomographic images obtained by imaging without using a three-dimensional image.
- V 4 ⁇ r 3/3 From this relationship, the radius r of a sphere having the same volume as the spheroid Sp can be calculated.
- This value r may be regarded as the radius of curvature, in which case the curvature is represented by (1 / r).
- FIG. 6A to FIG. 6C are diagrams showing an example of a debilitating spheroid.
- the viewing direction is set in a direction in which the spheroids are looked down slightly from above the side.
- FIG. 6A is an image (stereoscopic image) of a spheroid with relatively high viability, and a large number of cells gather to form a substantially spherical shape. However, there is a sign of collapse in the lower right part of the image.
- FIG. 6B is an image of a spheroid that has started to decay due to weakness
- FIG. 6C is an image of a spheroid that has further collapsed.
- the connection between the cells is weakened so that the spherical shape is not maintained, and the cells form irregular clusters of shapes.
- the characteristic amount such as the surface area and the volume, or the observation from above.
- Quantitatively it is possible to detect the collapse from the spherical shape by observing changes in the feature amount such as the curvature and sphericity of the spheroid surface.
- the surface of spheroid Sp (or its approximate curved surface) is not a perfect sphere. Therefore, in order to detect the collapse of the shape, it is effective to compare the curvatures seen from cross sections in two or more directions different from each other.
- the spheroid Sp in which the viability of the cell is lowered collapses so as to fall downward, that is, toward the bottom surface of the well W by the action of gravity. Therefore, it is considered that the curvature of the surface when the spheroid Sp is viewed from the horizontal direction changes particularly greatly.
- the spheroid Sp collapses. That is, it can be judged that the medicinal effect is appearing. When this is confirmed from the display image, it is effective to observe the spheroid Sp from a direction close to the horizontal direction.
- FIG. 7A to 7D are diagrams showing other examples of debilitating spheroids.
- FIG. 7A is an example of a stereoscopic image of another spheroid that has started to collapse.
- FIG. 7B is a cross-sectional view taken along the line AA in FIG. 7A, and corresponds to an image when the spheroid is viewed in the horizontal direction with the vertical plane as a cross section.
- the spheroid has a shape that is relatively close to a sphere, but granular objects are distributed around the spheroid on the well bottom surface Wb.
- FIG. 7C and 7D are a cross-sectional view taken along the line BB and a cross-sectional view taken along the line CC in FIG.
- the outer periphery of the spheroid has a shape that is relatively close to a circle.
- FIG. 7D viewed in a horizontal section closer to the well bottom surface Wb, an undefined and unclear image can be seen at a position surrounded by, for example, white circles around the spheroid that is a lump at the center. This is also an image of a granular material distributed on the well bottom surface Wb.
- an arc-shaped white area extending so as to surround the spheroid at a position away from the spheroid is a part of the curved well bottom surface Wb.
- the particulate matter distributed on the bottom surface Wb of the well is free cells deposited from the spheroids and deposited on the bottom of the well W or debris thereof.
- a phenomenon that occurs when a chemical substance reduces the viability of a cell there are cases where cells near the surface of the spheroid Sp cannot stay in that position and are released.
- the liberated cells have low activity and settle to the bottom of the culture solution and deposit on the well bottom surface Wb. Therefore, the presence / absence and amount of such free cells and debris can be used as effective information for indicating drug efficacy.
- the presence or absence of free cells or the like can be determined by visual observation from the image displayed on the display unit 352. Further, free cells and the like may be automatically detected by image processing.
- the presence or amount of free cells can be detected from the size and shape of the cell distribution range in an image showing a horizontal cross section close to the well bottom surface Wb as shown in FIG. 7D. This is because the cells gather in a relatively small area within the spheroid, whereas the free cells are scattered together.
- FIG. 8A to 8C are diagrams schematically showing a vertical section of a spheroid.
- FIG. 8A shows a spheroid Sp with high viability, and its vertical cross section is substantially circular.
- FIG. 8B shows a partially collapsed spheroid Sp, and the shape change accompanying the collapse appears remarkably in the lower part of the spheroid Sp.
- Z-axis direction Z-axis direction
- XY plane In observation from above (Z-axis direction) or in a horizontal section (XY plane), it is difficult to find such a change behind the spheroid.
- FIG. 8C shows a case where free cells and debris are precipitated on the well bottom surface Wb.
- the medicinal effect of the administered chemical substance appears, there are cases where many free cells D are generated from the spheroid Sp. Only by observing the shape of the spheroid Sp from the outside, such free cells D may be overlooked. In particular, in a two-dimensional captured image from above, it is difficult to discriminate between cells constituting the spheroid Sp and free cells D.
- the imaging unit 20 functions as the “image acquisition unit” of the present invention.
- the 3D restoration unit 33 and the feature amount calculation unit 34 function as the “stereoscopic image creation unit” and the “feature amount calculation unit” of the present invention, respectively.
- the well plate WP corresponds to the “container” of the present invention, and the holding unit 10 functions as the “holding means” of the present invention.
- the display unit 352 functions as the “display unit” of the present invention.
- each feature amount calculated in the above embodiment shows a part of an example as an index of the spheroid shape feature.
- the present invention is not limited to the use of these feature amounts. That is, only a part of the above-described feature amount may be used, or a feature amount other than the above may be used.
- the CPU 31, the 3D restoration unit 33, and the feature amount calculation unit 34 are individual functional blocks.
- the 3D restoration unit 33 and the feature amount calculation unit 34 may be configured by an integrated GPU (Graphic Processing Unit).
- achieved by one CPU may be sufficient.
- an optical coherence tomography (OCT) apparatus is used as the imaging unit 20 that performs tomographic imaging.
- OCT optical coherence tomography
- an imaging device based on another imaging principle that can perform tomographic imaging on a spheroid in a non-destructive manner for example, a confocal microscopic imaging device may be used as the “image acquisition unit” of the present invention.
- the optical coherence tomographic imaging apparatus as in this embodiment is advantageous in that imaging can be completed in a shorter time.
- the imaging apparatus is used as the “image acquisition unit” of the present invention, but the image processing apparatus according to the present invention itself does not necessarily have an imaging function. That is, an aspect in which tomographic image data captured by an external imaging device is received and only image processing is performed may be employed.
- the interface unit that receives image data from the outside functions as the “image acquisition unit” of the present invention.
- the present invention can be applied to a screening technique for finding a chemical substance having a medicinal effect on specific cells. Since it is possible to accurately evaluate the drug effect on a cell cluster in which cells are three-dimensionally assembled, it can be used for drug discovery that effectively acts in vivo.
- the present invention further includes a step of detecting free cells released from the cell clumps and deposited on the bottom surface of the container based on the tomographic image, and the feature value calculation result and the free cell detection result are obtained. It may be configured to determine the medicinal effect of the chemical substance based on it. Since dead cells detach from the cell clumps and accumulate on the bottom of the container, the presence of such free cells can be a strong evidence of the chemical efficacy of the chemical. Therefore, it is possible not only to pay attention only to the shape of the cell clump, but also to detect and evaluate the presence and amount of free cells deposited on the periphery, particularly the bottom of the container, so that the accuracy can be further improved.
- the present invention may be configured to acquire tomographic images for a plurality of different cross sections, for example. Since the shape of the cell cluster is not a perfect sphere, the evaluation accuracy can be further improved by performing the evaluation using a plurality of tomographic images in this way.
- a step of creating a three-dimensional image of the surface of the cell clump by image processing based on a plurality of tomographic images may be further provided.
- a pseudo-stereoscopic imaging of a cell cluster can be performed. If a three-dimensional image of a cell clump is created from a tomographic image, for example, the shape and surface state of the cell clump can be observed from various viewing directions. Thereby, comprehensive evaluation combined with the calculation result of the feature amount becomes possible, and the evaluation accuracy can be improved.
- the feature amount used in the present invention for example, at least one of the surface area of the cell clump, the volume of the cell clump, the curvature of the surface of the cell clump and the radius of curvature of the surface of the cell clump is used. Is possible. From the surface area and volume of the cell clump, the size of the cell clump can be known. The surface shape of the cell clump can be known from the curvature and radius of curvature of the cell clump surface. Any of these can be used as information for determining whether a cell clump is growing or debilitating.
- the present invention performs, for example, tomographic imaging of a cell clump a plurality of times at a predetermined time interval, and improves the efficacy of a chemical substance based on a temporal change in a feature amount obtained from a tomographic image acquired by each imaging. It may be configured to determine. Thus, it becomes possible to judge the medicinal effect of the said chemical substance more appropriately by investigating the change of the cell agglomeration with progress of time.
- a tomographic imaging technique capable of imaging non-contact and non-destructively on an imaging target, for example, an optical coherence tomographic imaging technique has been put into practical use. By applying this, imaging can be performed without affecting the cell agglomeration. Therefore, it is possible to observe the time change of the cell clump.
- the feature amount calculating means may be configured to calculate a feature amount with respect to an approximate curved surface corresponding to the surface of the cell clump obtained based on the stereoscopic image.
- a cell clump is a collection of a large number of cells, and irregular irregularities corresponding to the surface of each cell appear on the surface. Such fine irregularities do not represent the characteristics of the whole cell cluster. Therefore, by obtaining a feature amount by approximating it to a simpler curved surface, the shape feature of the cell clump can be quantified more accurately.
- the present invention further includes, for example, a holding unit that holds a container that holds a liquid containing cell clumps, and the image acquisition unit includes an imaging device that performs tomographic imaging of the cell clumps in the container. Also good.
- the tomographic image may be an image taken by an external imaging device, but the image processing device of the present invention includes a holding means for holding a container and an imaging device, so that a tomographic image optimal for the purpose of medicinal efficacy evaluation is provided. Can be obtained.
- an optical coherence tomographic imaging apparatus can be used as an imaging apparatus capable of performing such imaging.
- the present invention may be configured to include, for example, a display unit that has a function of displaying a stereoscopic image and can change the direction of the visual field with respect to the cell cluster in the display image. According to such a configuration, it is possible to provide the user with various information related to the appearance characteristics of the cell agglomeration, and the user can perform a comprehensive medicinal evaluation from the display image and the calculated feature amount. Can be done. While the invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense. Reference to the description of the invention, as well as other embodiments of the present invention, various modifications of the disclosed embodiments will become apparent to those skilled in the art. Accordingly, the appended claims are intended to include such modifications or embodiments without departing from the true scope of the invention.
- Image processing apparatus 10 Holding part (holding means) 20 Imaging unit (image acquisition means, optical interference imaging device) 21 Light source 22 Beam splitter 24 Reference mirror 25 Photo detector 30 Control unit 33 3D reconstruction unit (stereoscopic image creation means) 34 feature amount calculation unit (feature amount calculation means) 352 Display unit (display means) Sp spheroids (cell clumps) W well WP well plate (container)
Abstract
Description
関連出願の相互参照
以下に示す日本出願の明細書、図面および特許請求の範囲における開示内容は、参照によりその全内容が本書に組み入れられる:
特願2014-057594(2014年3月20日出願)。
この発明の前記ならびにその他の目的と新規な特徴は、添付図面を参照しながら次の詳細な説明を読めば、より完全に明らかとなるであろう。ただし、図面は専ら解説のためのものであって、この発明の範囲を限定するものではない。
z=ax+by+c … (式1)
が用いられる。点列(xi,yi,zi)、i=1~n(nは自然数)があるとき、上記(式1)にxi、yiを代入して得られたzの値とziとの差の二乗和が最小となるような定数a、b、cの値が求められる。具体的には、最小二乗和の式を定数a、b、cそれぞれを変数として偏微分した式の値を0とした方程式を連立させて解けばよい。
tGZ=tGG・X … (式4)
となり、未知数ベクトルXは次式:
X=(tGG)-1・tGZ … (式5)
と表すことができる。行列tGGは正規行列であり、正方行列となっている。(式5)の右辺については、例えばガウスの掃出し法を用いて解くことができる。
z=f(x,y)=ax2+by2+cxy+dx+ey+f … (式8)
とおき、6つの定数a~fを未知数とし、(式2)の係数行列Gに代えて、値1,xi,yi,xiyi,xi 2,yi 2を1行の要素とするn行6列の係数行列を用いて方程式を立てる。
V=4πr3/3
の関係から、スフェロイドSpと同じ体積を有する球の半径rが算出できる。この値rを曲率半径とみなしてもよく、その場合、曲率は(1/r)により表される。
また、画像処理によって自動的に遊離細胞等の検出が行われるようにしてもよい。例えば、図7Dに示すようなウェル底面Wbに近い水平断面を示す画像における細胞の分布範囲の大きさや形状などから、遊離細胞の有無やその量などを検出することができる。スフェロイド内では細胞が比較的小さな範囲に集まるのに対し、遊離細胞はまとまりなく散在するからである。
以上、特定の実施例に沿って発明を説明したが、この説明は限定的な意味で解釈されることを意図したものではない。発明の説明を参照すれば、本発明のその他の実施形態と同様に、開示された実施形態の様々な変形例が、この技術に精通した者に明らかとなるであろう。故に、添付の特許請求の範囲は、発明の真の範囲を逸脱しない範囲内で、当該変形例または実施形態を含むものと考えられる。
10 保持部(保持手段)
20 撮像ユニット(画像取得手段、光干渉撮像装置)
21 光源
22 ビームスプリッタ
24 基準ミラー
25 光検出器
30 制御ユニット
33 3D復元部(立体像作成手段)
34 特徴量算出部(特徴量算出手段)
352 表示部(表示手段)
Sp スフェロイド(細胞集塊)
W ウェル
WP ウェルプレート(容器)
Claims (12)
- 化学物質が細胞集塊に及ぼす薬効を評価する薬効評価方法において、
容器に担持された液体内に保持された前記細胞集塊を鉛直面と略一致する断面で断層撮像した、断層画像を取得する工程と、
前記断層画像に基づき前記細胞集塊の特徴量を算出する工程と、
前記特徴量の算出結果に基づいて、前記化学物質の薬効を判定する工程と
を備える薬効評価方法。 - 前記断層画像に基づき、前記細胞集塊から遊離して前記容器の底面に堆積した遊離細胞を検出する工程をさらに備え、
前記特徴量の算出結果と前記遊離細胞の検出結果とに基づいて前記化学物質の薬効を判定する請求項1に記載の薬効評価方法。 - 互いに異なる複数の断面について前記断層画像を取得する請求項1または2に記載の薬効評価方法。
- 複数の前記断層画像に基づく画像処理により、前記細胞集塊の表面の立体像を作成する工程を備える請求項1ないし3のいずれかに記載の薬効評価方法。
- 前記特徴量は、前記細胞集塊の表面積、前記細胞集塊の体積、前記細胞集塊の表面の曲率および前記細胞集塊の表面の曲率半径の少なくとも1つを含む請求項1ないし4のいずれかに記載の薬効評価方法。
- 前記特徴量は、互いに異なる複数の断面それぞれにおける前記細胞集塊の表面の曲率を含む請求項5に記載の薬効評価方法。
- 所定の時間間隔をおいて複数回、前記細胞集塊の断層撮像を行い、
各撮像で取得される前記断層画像から求められる前記特徴量の経時変化に基づいて、前記化学物質の薬効を判定する請求項1ないし6のいずれかに記載の薬効評価方法。 - 化学物質が細胞集塊に及ぼす薬効を評価する薬効評価のための画像処理装置において、
液体内に保持された前記細胞集塊を鉛直面と略一致する断面で断層撮像した、複数の断層画像を取得する画像取得手段と、
前記複数の断層画像に基づき、前記細胞集塊の立体像を作成する立体像作成手段と、
前記複数の断層画像または前記立体像に基づき、前記細胞集塊の特徴量を算出する特徴量算出手段と
を備える画像処理装置。 - 前記特徴量算出手段は、前記立体像に基づき求められた、前記細胞集塊の表面に対応する近似曲面について前記特徴量を算出する請求項8に記載の画像処理装置。
- 前記細胞集塊が含まれる前記液体を担持する容器を保持する保持手段をさらに備え、
前記画像取得手段は、前記容器内の前記細胞集塊を断層撮像する撮像部を有する請求項8または9に記載の画像処理装置。 - 前記撮像部は、光干渉断層撮像装置である請求項10に記載の画像処理装置。
- 前記立体像を表示する機能を有し、表示画像における前記細胞集塊に対する視野の方向を変更可能な表示手段を備える請求項8ないし11のいずれかに記載の画像処理装置。
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