JP6343935B2 - Growth information management system and growth information management program - Google Patents

Description

  The present invention relates to a system and a program for managing growth information of cells in a cell culture container (cells that require individual management such as a fertilized egg).

  The in vitro fertilized egg (zygote) is produced by fertilizing sperm and ovum in a culture system, and the fertilized egg goes through the cleavage, morula, and blastocyst stages, and then emerges from the zona pellucida It is possible to culture up to the blastocyst stage. Assistive reproductive technology (ART) to transfer a fertilized egg from the cleavage to the blastocyst stage to the uterus to give birth to a baby is not limited to the livestock region. Established in infertility medicine.

  However, the success rate of pregnancy by in vitro fertilization is not necessarily high. For example, in humans, the success rate of pregnancy is still about 25 to 35%. One of the causes is that the probability of obtaining a high-quality fertilized egg suitable for transplantation into the uterus in culture is not high. A cultured fertilized egg is individually observed with a microscope to determine whether it is a high-quality fertilized egg suitable for transplantation into the uterus.

  In in vitro fertilization, a microdrop method is often used in which a culture solution is dropped in a container, and a fertilized egg is placed in the container and cultured in vitro. Conventionally, in this microdrop method, a cell culture container having a single flat bottom surface and a diameter of 30 to 60 mm is used as a cell culture container, and a drop of the culture solution is placed on the bottom surface of the cell culture container. A method has been used in which a plurality of cells are prepared and cells are cultured therein.

  If a drop is created in a normal cell culture container, the position of the fertilized egg changes due to cell movement of the fertilized egg itself or convection in the drop, and it becomes difficult to identify the fertilized egg that has been cultured and observed in it. There was a problem. Therefore, a means for controlling the position of a fertilized egg has been demanded.

  In order to make the culture effect of fertilized eggs more efficient, it is considered preferable to use the interaction (paracrine effect) between fertilized eggs. In order to control the position of the fertilized egg while using these effects, a microwell having the same size as the fertilized egg is formed on the bottom surface of the cell culture container, and the culture solution is covered so as to cover a plurality of microwells. A system is known in which a drop is added and a fertilized egg is placed in a microwell filled with a culture solution and cultured. Thereby, while controlling the position of a plurality of fertilized eggs and enabling individual observation, a plurality of fertilized eggs can be cultured in a small amount of culture solution, and the paracrine effect can be utilized.

  On the other hand, in order to manage the growth state of individual fertilized eggs, it is necessary to identify individual microwells and to recognize the growth state of fertilized eggs in individual microwells. Conventionally, when managing growth information for each cell, such as a fertilized egg, a person distinguishes individual cells at a culture location and links them with growth information, or the feature amount is obtained from an image of the cell when observed under a microscope. Extraction was performed to identify and manage individual cells.

  However, human error is unavoidable with the above management method. For example, misidentification when a person distinguishes between individual microwells, or transcription errors in features detected by calculation (cell diameter, etc.) occur, and the individual microwells are identified and the cells in each microwell are identified. There was a risk of errors in association with growth information.

International Publication No. 2011/004568 Pamphlet

  The present invention provides a growth information management system and a growth information management program capable of associating microwells with cell growth information from an enlarged image of the microwells in a cell culture container having a plurality of microwells. With the goal.

In the cell culture container having a plurality of microwells and a plurality of first identifiers attached in pairs for each of these microwells, the present inventors have expanded specimens containing the first identifier and microwell pairs. It has been found that the above problem can be solved by acquiring an image and analyzing the first identifier and the feature amount of the cell in the enlarged specimen image.
That is, the present invention includes the following inventions.

(1) a cell culture container having a plurality of microwells and a plurality of first identifiers attached in pairs for each microwell;
Storage means for storing at least the correspondence between the enlarged specimen image including the pair of the first identifier and the microwell, the information on the first identifier, and the information on the feature amount of the cell in the microwell. ,
First identifier recognition means for recognizing the first identifier in the enlarged specimen image;
A feature amount calculating means for calculating a feature amount of a cell in the enlarged specimen image;
Registration means for registering at least the information on the enlarged specimen image, the information on the first identifier, and the information on the feature amount in association with each other;
A growth information management system.

(2) The feature amount includes the number of cells, cell shape, cell diameter, maximum length passing through the center of gravity of the cell region, circularity, cell aspect ratio, cell perimeter, cell area, cell The growth information management system according to (1), comprising at least one of a concentration, a uniformity of cells, a ratio of a predetermined cell to a whole cell, a cell arrangement, and a cell volume.

(3) The feature amount calculation means calculates a plurality of feature amounts of different cells in the enlarged specimen image, and calculates a predetermined score for evaluating the growth stage based on the plurality of feature amounts. The growth information management system according to 1) or (2).

(4) The feature amount calculation means determines whether the feature amount satisfies a predetermined condition using determination information for determining the growth stage of the cell, and determines the growth stage of the cell. The growth information management system according to any one of) to (3).

(5) The feature amount calculation means switches the determination information for determining the growth stage of the cell based on the elapsed time from the start of the culture of the cell, the feature amount, or the growth stage of the cell. The growth information management system according to (4).

(6) The registration unit registers the depth information when the enlarged specimen image is acquired in the storage unit in association with the enlarged specimen image, the information about the first identifier, and the information about the feature amount. The growth information management system according to any one of (1) to (5).

(7) A growth information management process for a cell culture container having a plurality of microwells and a plurality of first identifiers attached in pairs for each of the microwells. Information processing comprising at least computing means and storage means A program for causing a device to execute,
In the calculation means,
A first identifier recognition process for recognizing the first identifier in an enlarged specimen image including the pair of the first identifier and the microwell;
A feature amount calculation process for calculating a feature amount of a cell in the enlarged specimen image;
Registration processing for registering at least the information on the enlarged specimen image, the information on the first identifier, and the information on the feature amount in association with each other;
A program for running

(8) The feature amount includes the number of cells, cell shape, cell diameter, maximum length passing through the center of gravity of the cell region, circularity, cell aspect ratio, cell perimeter, cell area, cell The program according to (7), including at least one of a concentration, a uniformity of cells, a ratio of a predetermined cell to a whole cell, a cell arrangement, and a cell volume.

(9) The feature amount calculation processing calculates a plurality of feature amounts of different cells in the enlarged specimen image, and calculates a predetermined score for evaluating the growth stage based on the plurality of feature amounts. The program according to (7) or (8).

(10) The feature amount calculation processing includes determining whether the feature amount satisfies a predetermined condition using determination information for determining the growth stage of the cell, and determining the growth stage of the cell. , The program according to any one of (7) to (9).

(11) The feature amount calculation processing switches the determination information for determining the growth stage of the cell based on the elapsed time from the start of the cell culture, the feature amount, or the growth stage of the cell. The program according to (10), including:

(12) In the registration process, the depth information when the enlarged specimen image is acquired is registered in the storage unit in association with the enlarged specimen image, the information on the first identifier, and the information on the feature amount. The program according to any one of (7) to (11).

(13) An enlarged specimen image including a pair of the first identifier and the microwell from a cell culture container having a plurality of microwells and a plurality of first identifiers attached in pairs for each microwell. A first information processing apparatus comprising image acquisition means for acquiring;
A second information processing apparatus comprising storage means for storing the enlarged specimen image, information on the first identifier, and information on the feature amount of the cells in the microwell in association with each other;
With
The first information processing apparatus recognizes the first identifier in the enlarged specimen image, transmits information about the enlarged specimen image and the first identifier to the second information processing apparatus,
The second information processing apparatus receives information on the enlarged specimen image and the first identifier from the first information processing apparatus, calculates a feature amount of a cell in the enlarged specimen image, and the enlarged specimen image A growth information management system that registers at least the information about the first identifier and the information about the feature amount in association with each other.

  According to the present invention, in a cell culture container having a plurality of microwells, the microwells can be associated with cell growth information from an enlarged image of the microwells.

It is a block diagram of one Embodiment of the growth information management system of this invention. It is the schematic which shows the upper side figure of one Embodiment of the cell culture container of this invention. It is the schematic which shows the enlarged top view of the cell accommodating part of one Embodiment of the cell culture container of this invention. It is the schematic which shows the enlarged top view of the cell accommodating part of one Embodiment of the cell culture container of this invention. It is the schematic which shows the enlarged top view of the cell accommodating part of one Embodiment of the cell culture container of this invention. It is a conceptual diagram of the enlarged image which image | photographed the pair of the microwell and the identifier with the microscope. It is the schematic which shows one Embodiment of the sample information database of this invention. It is the schematic which shows one Embodiment of the 1st identifier corresponding | compatible table of this invention. It is the schematic which shows one Embodiment of the 2nd identifier corresponding | compatible table of this invention. It is the schematic which shows one Embodiment of the growth information database of this invention. It is the schematic which shows one Embodiment of the determination profile table of this invention. It is a conceptual diagram explaining one Embodiment of the outline extraction process of this invention. It is a conceptual diagram explaining the processing content of one Embodiment of the 1st identifier recognition process part of this invention. It is a conceptual diagram explaining the processing content of one Embodiment of the 1st identifier recognition process part of this invention. It is a conceptual diagram explaining the processing content of one Embodiment of the feature-value calculation process part of this invention. It is a conceptual diagram explaining the processing content of one Embodiment of the feature-value calculation process part of this invention. It is a figure which shows a blastocyst simply. It is a figure explaining the morphological evaluation of an inner cell mass and trophectoderm. It is a figure explaining the morphological evaluation of a pronuclear stage embryo. It is a figure explaining switching of the profile according to elapsed time. It is a flowchart explaining the flow of a process of one Embodiment of the growth information management system of this invention. It is a flowchart explaining the flow of the feature-value calculation process of the growth information management system of this invention. It is a figure which shows another embodiment of a growth information database.

  Hereinafter, examples in which the present invention is applied to a human fertilized egg will be described. For example, when cultivating a human fertilized egg, the growth stage of the fertilized egg is usually determined while culturing, thereby determining whether the fertilized egg is of good quality suitable for transplantation into the uterus. . The growth information management system of the present invention realizes management of growth information without error by associating the microwell with the growth information of the cell from the enlarged image of the microwell, and automatically calculates the feature quantity of the fertilized egg. It is possible to determine the growth stage of the fertilized egg.

  FIG. 1 shows a configuration diagram of an embodiment of the growth information management system of the present invention. The growth information management system 1 includes a cell culture container 100, an image acquisition device 110, a second identifier reading device 120, and a growth information management device 130. Hereinafter, each component of the growth information management system 1 will be described.

  As shown in FIG. 1, the cell culture container 100 has a first identifier 101 and a second identifier 102. 2 and 3 show a specific configuration of the cell culture container 100. FIG. The cell culture container 100 of this embodiment has a bottom portion 103 and a side wall 104, and has a cell storage portion 106 in which a plurality of microwells 105 for storing cells are arranged. The shape of the bottom portion 103 is not particularly limited, and may be a polygonal shape such as a triangle and a quadrangle, or may be a circular shape (including a circle, a substantially circular shape, an elliptical shape, and a substantially elliptical shape), and the side wall 104 is an outer edge of the bottom portion 103. It is formed to surround.

  Usually, the side opposite the bottom 103 is open, and the shape of the opening is preferably the same as the shape of the bottom 103. Preferably, those having a circular opening and an opening width of preferably 30 to 60 mm, particularly 35 mm are used. This is the same size as a conventional cell culture vessel used for cell culture, can be easily produced from a general-purpose cell culture vessel, and is easy to adapt to existing culture devices. Are preferred. In addition, the cell culture container 100 may have a lid | cover similarly to a normal cell culture container.

  3 and 4 are schematic views showing an enlarged top view of the cell accommodating portion of one embodiment of the cell culture container of the present invention. In the vicinity of the plurality of microwells 105, the first identifier 101 is attached in pairs for each microwell 105, and the relative position of the first identifier 101 with respect to the paired microwell 105 is the first. It is characterized by being different for each pair of the identifier 101 and the microwell 105. Since the relative position of the first identifier 101 with respect to the microwell 105 is different for each microwell 105, only one pair of the microwell 105 and the first identifier 101 is observed, so that the microwell 105 has a plurality of microwells 105. The position can be specified. Since the first identifier 101 is attached in the vicinity of each microwell 105, when observing at a high magnification, it is not necessary to greatly shift the observation position from the microwell 105, and quick observation is possible. Furthermore, even when the cells are photographed at a high magnification, the first identifier 101 is photographed together with the microwell 105 in the magnified specimen image, so there is no need to manually add information to the magnified specimen image, Complicated work can be avoided, and the risk of an association error due to an operator's mistake can also be avoided.

  The microwell 105 forms a recess having a wall surface and an opening, and may be a recess provided directly as a recess in the bottom 103 of the cell culture vessel 100 or a recess formed by a member protruding from the bottom 103. Therefore, the area of the opening of each microwell 105 in the top view is, in other words, the area of the figure formed by the outer edge of the opening of the microwell 105. The figure formed by the outer edge of the opening of the microwell 105 is not particularly limited, and is a polygonal shape such as a triangle and a quadrangle, a circle (including a circle, a substantially circle, an ellipse, and a substantially ellipse), or U Although the shape of a character etc. may be sufficient, Preferably it is circular. Note that the microwell 105 may have a shape in which a stepped concave portion is formed toward the center of the microwell 105. This is because cells and the like easily flow to the center position.

When the outer edge of the opening of the microwell 105 is circular, the opening width is equal to the diameter of the circle (R in FIG. 3), and the diameter is larger than the maximum dimension of the cells to be cultured. Note that the area of the opening of each microwell 105 in the top view of the cell culture container 100 is preferably 3 mm ² or less, more preferably 1 mm ² or less, still more preferably 0.5 mm ² or less, and preferably 0.03 mm. ² or more.

  In the bottom 103 of the cell culture vessel 100 of the present invention, the number of microwells 105 is preferably 4 or more, more preferably 8 or more, for example 10 or more, preferably 50 or less, more preferably 30 or less. Therefore, a plurality of cells can be cultured by placing one cell such as a fertilized egg in one microwell 105 one by one.

  The plurality of adjacent microwells 105 are preferably arranged in a square lattice shape or a close-packed shape. For example, 25 microwells can be arranged in a 5 × 5 square lattice. By arranging in a square lattice pattern or in a close-packed pattern, the position of each microwell 105 in the bottom 103 of the cell culture vessel 100 can be more easily identified in combination with the first identifier 101, and can be applied to automated processing. Cheap.

  The arrangement of the plurality of microwells 105 may be an arrangement in which a part of the arrangement is omitted from a square lattice or a close-packed arrangement. For example, a case where eight or more microwells are arranged at equal pitches on the sides and vertices of the parallelogram to form a cell accommodation unit. Parallelograms include squares, rectangles, rhombuses and other parallelograms. The microwells 105 are arranged on the sides and vertices of the parallelogram means that the center of gravity of the figure formed by the outer edge of the opening of the microwell 105 is arranged on the sides and vertices of the parallelogram. Sure. For example, in the embodiment shown in FIG. 3, eight microwells are arranged one by one at the four vertices of the square and one at the midpoint of the four sides.

  The position of the first identifier 101 attached in a pair with each microwell 105 may be inside or outside the microwell 105, but is preferably arranged outside the microwell 105. This is because if it is provided inside the microwell 105, the observation of the fertilized egg may be hindered and the culture performance of the fertilized egg may be affected. Preferably, as shown in FIG. 3 and FIG. 4, the first identifier 101 is attached to a gap between a plurality of arranged microwells 105. The first identifier 101 is sufficiently small so that it can be placed in such a gap. The size of the first identifier 101 is preferably smaller than the size of the microwell 105.

  In addition, the first identifier 101 is attached sufficiently close to the paired microwell 105 so that it is clear which microwell 105 is paired with. Accordingly, each first identifier 101 is preferably attached so that the distance from the paired microwells 105 is the smallest among all the microwells 105. The distance between the first identifier 101 and the microwell 105 is defined as the distance between the centroid of the graphic formed by the opening of the microwell 105 and the centroid of the graphic formed by the first identifier 101. Therefore, the distance between the first identifier 101 and the microwell 105 is preferably larger than ½ of the opening width of the microwell 105 and smaller than the pitch between the microwells 105.

  The first identifier 101 is preferably attached to all of the microwells 105 in the cell storage unit 106, but several microwells 105 to which the first identifier 101 is not attached (for example, included in the cell storage unit 106). 10% or less of the total number of microphone wells). This is because the first identifier 101 is not necessary in such a microwell 105 when cells are not accommodated and there is a microwell 105 that is not an observation target. One first identifier 101 is preferably attached to the paired microwells 105, but two or more identifiers may be attached.

  The shape of the first identifier 101, that is, the shape of the graphic formed by the first identifier 101 is not particularly limited. Examples of figures include figures such as letters, numbers, polygons, arrows, lines (bars), dots, QR codes, barcodes, and combinations thereof. Since the first identifier 101, which is preferably smaller in size than the microwell 105, is attached in the vicinity of the microwell 105 suitable for individually containing cells such as fertilized eggs, the shape of the first identifier 101 is A simple shape that is easy to mold is preferred. This is because the cell culture container 100 is often manufactured by injection molding, so that it is difficult to form a very complicated shape with a minute size. Even if the shape of the first identifier 101 is simple, that is, even if the information of the first identifier 101 itself is small, the position of each microwell 105 is specified by adding information on the relative position to the microwell 105 can do. In addition, if the first identifier 101 has a complicated shape, the yield in manufacturing the cell culture container 100 may decrease. However, by using a simple shape, a decrease in yield can be avoided and the manufacturing cost can be reduced. Can do.

  Therefore, the first identifier 101 preferably has a dot shape or a linear shape (bar shape). 3 and 4 are examples of the dot-shaped first identifier 101. FIG. In this example, eight microwells 105 are arranged one by one at four vertices of a square and one by one at the midpoints of the four sides to constitute the cell storage unit 106. One dot-like first identifier 101 is attached in the vicinity of each microwell 105. The relative position of the first identifier 101 with respect to the paired microwell 105 is different for each pair of the first identifier 101 and the microwell 105.

  The relative position of the first identifier 101 with respect to the paired microwell 105 is different for each pair of the first identifier 101 and the microwell 105. The distance between the first identifier 101 and the paired microwell 105 is , Different for each pair, and different angles of the first identifier 101 to the paired microwell 105.

  The distance between the first identifier 101 and the microwell 105 is as described above, but the angle α of the first identifier 101 with respect to the microwell 105 can be defined as follows. For example, in the embodiment shown in FIG. 3 and FIG. 4, the angle α is a straight line parallel to the straight line X and the microwell 105 when a single straight line X is drawn on the bottom 103 of the cell culture container 100 in a top view. And the straight line Y passing through the center of gravity of the first identifier 101 (FIG. 4). And the position of the specific microwell 105 in the plurality of microwells 105 can be specified by arranging the angle α to be different for each pair of the microwell 105 and the first identifier 101. The amount of information can be increased by combining distance and angle. In this embodiment, since the angle α of the first identifier 101 with respect to the microwell 105 is different for each pair, the position of each microwell 105 is specified only by observing the pair of microwells 105 and the first identifier 101. be able to.

  5 and 6 are examples of the linear first identifier 101. FIG. In this example, eight microwells 105 are arranged one by one at four vertices of a square and one by one at the midpoints of the four sides to constitute the cell storage unit 106. One linear first identifier 101 is attached in the vicinity of each microwell 105.

  FIG. 6 shows a microscopic image of a pair of the microwell 105 and the first identifier 101 at positions A, E, and H in FIG. In this embodiment, the distance between the first identifier 101 and the microwell 105 is substantially the same for any pair, but the angle α of the first identifier 101 with respect to the microwell 105 and / or the length of the line (bar). However, since each pair is different, the position of each microwell 105 can be specified only by observing the pair of microwells 105 and the first identifier 101. Further, since each first identifier 101 is linear in the same direction and attached to the right side with respect to the microwell 105, the microwell 105 photographed at a high magnification and the first identifier 101 are taken. Also in the pair of enlarged images, the orientation of the cell culture container at the time of imaging can be specified based on the position of the first identifier 101 and the direction of the line (bar). The “direction” here is a rotation angle and is different from the angle α. For example, when the dots are replaced with lines (bars) in the embodiment of FIG. 4, it refers to the angle at which the line rotates (spins) with respect to the straight line X at the dot position, that is, the angle formed by the straight line X and the identifier line. Using this amount of orientation information, the orientation of the cell culture vessel can be specified.

  In some cases, it is preferable that the plurality of first identifiers 101 attached to the plurality of microwells 105 are arranged on the same axis. Here, the plurality of microwells 105 do not have to be all the microwells 105 in the cell accommodating portion 106, preferably 2 or more, more preferably 3 or more, and still more preferably 4 or more of them. A plurality of microwells on the same axis. The plurality of first identifiers 101 attached to the plurality of microwells 105 whose centroids are on the same axis are arranged on the same axis, thereby changing the microwell 105 to be observed in high magnification observation. In this case, since the pair of the microwell 105 and the first identifier 101 can be captured by moving the cell culture container 100 only in the X-axis or Y-axis direction with respect to the lens, rapid observation becomes possible.

  Next, the second identifier 102 will be described. A second identifier 102 is attached to the cell culture container 100. Examples of the second identifier 102 include characters, numbers, figures such as polygons, QR codes, barcodes, IC tags, dot patterns (coded patterns), and combinations thereof. Here, the dot pattern (coded pattern) technique will be briefly described. For example, the dot pattern is printed on the surface of a dedicated paper for an electronic pen. The dot pattern is a combination of vertical and horizontal 6 × 6 = 36 dots arranged on a lattice with an interval of about 0.3 mm. The vertical and horizontal 6 × 6 dots are arranged so as to form a unique pattern no matter which portion of the dedicated paper is taken 6 × 6 dots. A dot pattern formed by these 36 dots holds position coordinates (for example, on which position the dot pattern is located on the dedicated paper) and a dot pattern address that is a unique identifier for each dedicated paper. . Each dot is associated with predetermined information by the shift direction from the reference position of the lattice. That is, according to this technique, the dot pattern can be made to correspond to the second identifier 102 by making the shift direction from the reference position of the lattice of each dot correspond to characters, numbers, and the like. The dot pattern can be photographed by reading means such as an electronic pen with a built-in camera. The dot pattern technique described here is also described in Japanese Patent Application Laid-Open Nos. 2004-252607 and 2004-054375, and the techniques described in these documents can be used. The second identifier 102 may be attached to the side wall 104 or the bottom 103 of the cell culture container 100. In addition, when the cell culture container 100 includes a lid, the second identifier 102 may be attached to the lid. The second identifier 102 is different for each cell culture container 100, and the cell culture container 100 can be specified by recognizing the second identifier 102.

  When one pair of the microwell 105 and the first identifier 101 is photographed at a high magnification and a plurality of pairs are photographed by repeating this, it is necessary to always make the orientation of the cell culture container 100 constant when photographing. Otherwise, the angle α includes the inclination of the cell culture container 100 itself, and the relative position may not be distinguished on the enlarged image even though the relative position is different on the cell culture container 100. Because there is. From the viewpoint of always keeping the orientation of the cell culture container 100 at the time of photographing, the cell culture container 100 is used to specify the orientation of the cell culture container 100 different from the first identifier 101 and the second identifier 102. It is preferable to attach a third identifier.

  Since the third identifier is used when photographing the enlarged specimen image, it is preferable that the third identifier can be visually confirmed. Therefore, in the top view of the cell culture container 100, the area of the third identifier is preferably larger than the area of the opening of the microwell 105. The third identifier is preferably attached to the outside of the cell storage unit 106 on the bottom portion 103 where a plurality of microwells 105 are arranged (for example, 107 in FIG. 2). The third identifier may be attached to the side wall 104 of the cell culture container 100. Alternatively, the orientation of the cell culture container can always be constant depending on the shape of the cell culture container 100 itself. That is, by making the outer peripheral shape of the side wall 104 of the cell culture container 100 a shape that can specify the orientation of the cell culture container, for example, a shape lacking a circle, the orientation of the cell culture container 100 at the time of photographing is always constant. Is also possible. In this case, the shape of the cell culture container is not particularly limited as long as the orientation of the cell culture container 100 can be specified.

  Alternatively, if the first identifier 101 is linear, unlike the dot shape, it has two-dimensional information. Therefore, even if there is no third identifier, the microwell 105 and the first identifier captured at a high magnification are used. In the enlarged image of 101 pairs, the orientation of the cell culture container can be specified to some extent. That is, each of the first identifiers 101 has a linear shape facing in the same direction in the top view of the cell culture container 100, so that an enlarged image of a pair of the microwell 105 and the first identifier 101 photographed at a high magnification. Also, the direction of the cell culture container at the time of photographing can be specified to some extent based on the direction of the line (bar).

  In addition, for all the plurality of microwells 105 in the cell storage unit 106, the first identifier 101 is attached only to the right side, only the left side, only the upper side, or only the lower side of the microwell, so Also in the enlarged image of the pair 105 and the first identifier 101, the orientation of the cell culture container 100 can be specified. Since the approximate position of the first identifier 101 is determined to some extent for each target microwell 105, it is easy to determine the imaging position when manually imaging a fertilized egg at a high magnification. Here, the right side, the left side, the upper side, or the lower side of the microwell can be defined as respective ranges obtained by dividing 360 ° around the center of gravity of the microwell into four parts. For example, α shown in FIG. A range of ˜135 ° can be defined as the upper side. Therefore, even when the identifier is attached only to the upper side of the microwell, it is possible to make the relative position different for each microwell within the range.

  Next, other components of the growth information management system 1 will be described. The image acquisition device 110 captures an enlarged specimen image including cells in the microwell 105 of the cell culture container 100 and the first identifier 101, and is, for example, a microscope. As the image acquisition device 110, for example, a device equipped with a 1/2 inch CCD element, 4, 10, and 20 times objective lens is often used.

  The second identifier reading device 120 is a device that reads the second identifier 102 of the cell culture container 100. As the second identifier reading device 120, a device corresponding to the second identifier 102 may be employed, and examples thereof include a known character / graphic recognition device, a camera, a barcode scanner, and an IC tag reader.

  The growth information management device 130 is configured by an information processing device such as a personal computer or a workstation. This information processing apparatus includes a processor such as a central processing unit (CPU), a storage device 131 such as a memory and a hard disk, an input unit 132 such as a keyboard and a mouse, and an output unit 133 such as a display. I have. In FIG. 1, the growth information management device 130 is shown as one information processing device, but the present invention is not limited to this. The constituent elements of the growth information management apparatus 130 may be distributed to a plurality of information processing apparatuses on the network. Various tables and databases may be stored in other information processing apparatuses or servers on a network.

  Next, various information used in the growth information management system 1 of the present embodiment will be described. The storage device 131 stores a specimen information database 141, well design information 142, a second identifier correspondence table 143, a growth information database 144, and a determination profile table 145. These various tables and databases will be described using a “table” structure in the following description, but they may not necessarily be represented by a data structure using tables, but may be represented by other data structures. Therefore, in order to show that the data structure does not depend on the data, hereinafter, various tables and databases may be simply referred to as “information”.

  FIG. 7 is an example of the sample information database 141. The specimen information database 141 stores various types of information related to cells in the cell culture container (cells that require individual management such as fertilized eggs). The specimen information database 141 includes a patient ID 701, a treatment date and time 702, a cell culture container ID (container ID) 703, and a well ID 704 as configuration items. The patient ID 701 is a number that uniquely identifies a patient. By associating the patient ID 701 with the cell culture container ID 703 and the well ID 704, it can be applied to ID management such as in the case of human infertility treatment. The treatment date and time 702 is a string of numbers indicating the treatment date and time. Although mentioned later, when determining the growth stage of a fertilized egg, it may determine based on the elapsed time from the operation date. In such a case, it is preferable to manage reference information such as the treatment date and time in the specimen information database 141. The cell culture container ID 703 is a number that uniquely identifies the cell culture container 100. The well ID 704 is an ID for identifying the position of each microwell 105 in the cell culture container 100. By using the well ID 704, it is possible to manage information on a specimen of a cell (eg, a fertilized egg) arranged inside the microwell 105.

  The well design information 142 includes at least a first identifier correspondence table. FIG. 8 is an example of a first identifier correspondence table. The first identifier correspondence table includes at least a well ID 801 and information 802 indicating the relative position between the first identifier 101 and the microwell 105 (here, the angle α of the first identifier 101 with respect to the microwell 105) as configuration items. . Therefore, the position (well ID 1002) of each microwell 105 can be specified only by observing the pair of microwells 105 and the first identifier 101. In this embodiment, the well ID 801 is used as information regarding the position of the microwell. However, the present invention is not limited to this, and other forms are possible as long as the information can identify the position of the microwell.

  In the first identifier correspondence table, the value of the angle α may be set with a predetermined width. This is because an error may occur in image processing in the growth information management apparatus 130 described later. 8 shows only the information on the angle α, but when the first identifier 101 is a line (bar), information (angle α) indicating the relative position between the first identifier 101 and the microwell 105 and / or Alternatively, information (such as a line length and direction) included in the first identifier 101 itself may be stored. As described above, examples of the first identifier 101 include characters, figures, polygonal shapes, arrows, lines (bars), dots, QR codes, barcodes, and combinations thereof. Therefore, the first identifier correspondence table only needs to store information related to the first identifier 101 corresponding thereto. In the above description, one first identifier correspondence table is shown. However, when the number or arrangement (alignment) of the first identifiers 101 changes for each cell culture container, the first identifier correspondence is made for each type of cell culture container. You may hold a table.

  In addition to the first identifier correspondence table, the well design information 142 includes, for example, the shape of the microwell 105, the dimension of the outer edge of the opening of the microwell 105, the area of the opening of the microwell 105, and the space between the microwells 105. Design information such as pitch may be included. The well design information 142 may include design information of the first identifier 101 that is paired with each microwell 105. The design information of the first identifier 101 is information such as the shape, size, and position of the first identifier 101.

  FIG. 9 is an example of the second identifier correspondence table 143. The second identifier correspondence table 143 includes at least a cell culture container ID 901, second identifier information 902, and a container type 903 as configuration items. Using this table, each cell culture container 100 can be identified from the second identifier 102 attached to the cell culture container 100. The container type 903 is information indicating the type of cell culture container. As described above, the number and arrangement of the first identifiers may change for each cell culture container. Therefore, by having information on the container type 903, the first identifier correspondence table corresponding to the container type 903 can be selected.

  FIG. 10 is an example of the growth information database 144. The growth information database 144 stores an enlarged sample image acquired from the image acquisition device 110 and various types of information related to this image. The growth information database 144 includes a cell culture container ID 1001, a well ID 1002, and growth information 1003 as constituent items. Thus, in the growth information database 144, the growth information 1003 is associated with the cell culture container ID 1001 and the well ID 1002. Growth information 1003 includes image information 1004, information 1005 indicating whether or not a fertilized egg exists in the well in the image, information 1006 indicating the first feature amount of the cell in the well in the image, It includes information 1007 indicating the second feature amount of the cells in the well, and date and time 1008 when the image information is acquired. Information 1006 indicating the first feature amount is information on the diameter of the blast ball, and information 1007 indicating the second feature amount is information on the number of blast balls. In this example, information about two feature amounts is managed for the image information 1004. However, the present invention is not limited to this example, and one or more feature amounts may be managed. Also, the feature amount is not limited to the example of FIG. 10, and various types of feature amount will be described later. Furthermore, as growth information, information indicating the growth stage of cells (grade information of classification such as Veek, Gardner, etc.) may be stored together with the feature amount. Here, the cell growth stage means the growth stage of one whole cell, and in the following, the growth stage of one fertilized egg will be described as an example.

  Therefore, by referring to the growth information database 144, each image information 1004 corresponds to an enlarged specimen image of any microwell, and information on the growth stage of cells in the microwell can be managed simultaneously. In addition, by referring to both the growth information database 144 and the sample information database 141, each image information 1004 corresponds to which microwell enlarged sample image and which patient's time and date of treatment. Can manage. In the present embodiment, the growth information database 144 is created. However, an item in which image information and growth information can be registered in the sample information database 141, and an enlarged sample image and growth information are registered in the sample information database 141. But you can.

  FIG. 11 is an example of the determination profile table 145. The determination profile table 145 stores determination information for determining a cell growth stage. The determination profile table 145 includes an application time 1101, a grade 1102, and a condition 1103 as configuration items. Although mentioned later, when determining the growth stage of a fertilized egg, it may determine based on the elapsed time from the operation date. The application time 1101 indicates the elapsed time from the treatment date (culture start date). The condition 1103 stores information on the feature amount that should be calculated from the enlarged specimen image at the time of application 1101 and information on the condition that the feature amount should satisfy. Thereby, according to the elapsed time from the operation date and time, it is possible to switch the characteristic amount of the cell to be calculated and the determination condition for determining the growth state of the fertilized egg. For example, in the example of FIG. 11, when the elapsed time is 2 to 3 days, the profile of the first record considering the evaluation of Veek is selected. When the elapsed time is 4 to 5 days, the profile of the second record in consideration of Gardner's evaluation is selected. Details of Veek and Gardner will be described later. Grade information when the condition 1103 is satisfied is stored in the grade 1102. Using this grade 1102 information, it becomes possible to determine the growth stage of fertilized eggs (or the quality of fertilized eggs).

  Next, the growth information management device 130 will be described in detail. As illustrated in FIG. 1, the growth information management apparatus 130 includes a first identifier recognition processing unit 151, a second identifier recognition processing unit 152, a feature amount calculation processing unit 153, and a database creation processing unit 154. The growth information management apparatus 130 analyzes the enlarged specimen image photographed by the image acquisition apparatus 110 using these processing units 151 to 154, and obtains the enlarged specimen image information, the well ID, and the growth information based on the various tables described above. Associate. This makes it possible to uniquely identify the growth information for the microwell.

  In this embodiment, you may implement | achieve each process part 151-154 of the growth information management apparatus 130 as a function of the program run on a computer. That is, the processing units 151 to 154 may be stored in the memory as program codes, and the processing units 151 to 154 may be realized by the CPU executing the program codes. The processing units 151 to 154 will be described below.

  The first identifier recognition processing unit 151 receives an enlarged specimen image including the cells in the microwell 105 of the cell culture container 100 and the first identifier 101 from the image acquisition device 110, and executes an outline extraction process on the enlarged specimen image. To do. A technique known to those skilled in the art can be applied as the contour extraction process. As an example of the contour line extraction process, there is a process of executing a binarization process for converting an enlarged specimen image into a two-tone image of white and black and extracting a contour line from the binarized image. . In general, since the object to be observed is transparent and it is difficult to add a contrast to the boundary, after performing a differential operation on the change of the pixel value, a process of converting to a two-tone image may be executed. . Moreover, as another outline extraction process, you may use the technique as described in Unexamined-Japanese-Patent No. 2011-192109. In this document, the outline of a fertilized egg is determined by creating a reference profile and a candidate profile, and calculating the degree of similarity indicating the similarity between the reference profile and the candidate profile. You may apply to determination of the outline of a line or a 1st identifier. The first identifier recognition processing unit 151 forms the contour of the figure formed by the outer edge of the opening of the microwell 105 (hereinafter referred to as “outer contour”) and the first identifier 101 by the contour extraction process. The contour line of the figure is extracted, and a contour line extracted image is output (FIG. 12).

  Further, interpolation processing and / or region definition processing may be executed after the contour line extraction processing in the first identifier recognition processing unit 151. Interpolation processing is processing that estimates and interpolates a portion where the contour line is interrupted by combining a pattern prepared in advance and the contour line extraction image. For example, if only the outline extraction process is performed, the outline of the microwell 105 and the outline of the figure formed by the first identifier 101 may be interrupted. Therefore, the pattern of the shape of the microwell 105 and the pattern of the shape of the first identifier 101 are registered in advance, and the respective contour lines are interpolated by superimposing the respective patterns on the contour line extraction image. The area definition process is a process for defining an area surrounded by an outline. For example, when the contour line on the contour line extracted image is discontinuous, a region surrounded by the contour line interpolated after the contour line interpolation process is defined. By performing this region definition process, it is possible to determine a region for pattern matching to be performed later and a region for calculating the size or area of the region surrounded by the outline.

  The first identifier recognition processing unit 151 performs pattern detection processing on the contour line extracted image. Here, the pattern detection processing is to detect which contour line is the outer peripheral contour line of the microwell 105 in the contour line extraction image and which contour line is the contour line of the first identifier 101. is there. As the pattern detection process, a technique known to those skilled in the art can be applied. As an example, there is a method in which a template having the shape of the microwell 105 and a template having the shape of the first identifier 101 are stored in advance in the growth information management device 130, and pattern matching is performed using each template. The first identifier recognition processing unit 151 detects a portion having a predetermined similarity or higher with respect to each template as the contour line of the microwell 105 or the contour line of the first identifier 101. Note that the template may be appropriately enlarged / reduced according to the enlargement magnification of the image acquisition device 110.

  The processing for detecting the outer contour of the microwell 105 and the contour of the first identifier 101 in the contour extraction image is not limited to this, and other methods may be used. For example, the detection may be performed using the design information of the microwell 105 and the design information of the first identifier 101 in the well design information 142. For example, by comparing information such as the size and area of the figure formed by the contour line on the contour line extracted image with the design information of the microwell 105 and the design information of the first identifier 101, the outer contour line of the microwell 105 The contour line of the first identifier 101 may be specified.

  The 1st identifier recognition process part 151 performs the recognition process of the 1st identifier 101 with respect to an outline extraction image. The first identifier recognition processing unit 151 calculates an angle α of the first identifier 101 with respect to the microwell (FIG. 13). When the first identifier 101 is a line (bar), the first identifier recognition processing unit 151 may calculate the angle α and / or the length of the line (bar). Thereafter, the first identifier recognition processing unit 151 refers to the first identifier correspondence table by the calculated angle α and outputs a well ID corresponding to the calculated angle α.

  In addition, the process of the 1st identifier recognition process part 151 is not limited to this, Another method may be sufficient. In the processing of the first identifier recognition processing unit 151, it is only necessary that the microwell 105 and the first identifier 101 can be associated with the well ID based on the relative positional relationship between the microwell 105 and the first identifier 101. As shown in FIG. A plurality of templates including the above may be prepared and template matching may be performed. If each template and the well ID are associated with each other in the first identifier correspondence table, the enlarged specimen image and the well ID can be associated from the result of template matching.

  The second identifier recognition processing unit 152 refers to the second identifier correspondence table 143 based on the information of the second identifier 102 read by the second identifier reader 120, and outputs the cell culture container ID corresponding to the information. In addition to this method, a method may be used in which the cell culture container ID is recognized in advance by another method, and the second identifier 102 is read by the second identifier reader 120 to check the recognition accuracy.

  The feature amount calculation processing unit 153 first determines whether there is a cell in the microwell 105 from the enlarged specimen image. For example, the diagram on the left side of FIG. 12 (enlarged specimen image before contour extraction) shows a case where cells exist in the microwell 105. When cells are not present in the microwell 105, the pixel values in the microwell 105 are uniformly distributed in the enlarged specimen image. For example, the feature amount calculation processing unit 153 determines whether there is a change in the pixel value in the microwell 105 and determines whether there is a cell in the microwell 105. This determination process is an example, and other known methods may be used as long as the presence or absence of cells is determined. The determination result here can be registered as information 1005 in the growth information database 144.

  In addition, the feature amount calculation processing unit 153 calculates various feature amounts from the image information of the enlarged specimen image. Below, the feature-value of a human fertilized egg is demonstrated as an example. When cultivating a human fertilized egg, the growth state of the fertilized egg is usually determined in stages after fertilization, thereby determining whether the fertilized egg is of good quality suitable for uterus transplantation.

  First, morphological evaluation performed at the time of the second to third days after the start of culture will be described. There is a method for evaluating the grade of embryos at the time of 3-8 cell stage embryos 2-3 days after fertilization. In this evaluation, the Veek classification is generally used. In the Veek classification, embryos with equally divided cells and less fragmentation (cell debris) are considered better.

The grades of Veek are shown below.
Grade 1: Embryo with uniform cell (blastomere) morphology and no fragmentation Grade 2: Embryo with uniform cell (blastomere) morphology but no fragmentation Grade 3: Cell (blastomere) Embryos with heterogeneous morphology. Or embryos that accept a small amount of fragmentation.
Grade 4: Embryos with uniform or non-uniform cell (blastomere) morphology and significant fragmentation.
Grade 5: Embryo with few cells (blastomere) and marked fragmentation.

  Next, the feature amount calculation processing by the feature amount calculation processing unit 153 with reference to the Veek classification will be described. FIG. 15 shows an example of calculating the degree of uniformity of cells (blastomere) from the enlarged specimen image. In FIG. 15, the enlarged specimen image is shown by a simple illustration. Note that the subject of the processing described below is a feature amount calculation processing unit 153.

  FIG. 15A shows a cell portion cut out from the enlarged specimen image for easy understanding of the present technique. Note that the feature amount calculation processing unit 153 may perform image processing of the enlarged specimen image as it is, or may perform image processing by cutting out only a cell portion of the enlarged specimen image. As described above, the pixel value of the cell portion in the microwell 105 varies, and an edge can be detected by a known method, so that an image of the cell portion can be cut out. First, an edge corresponding to the outline of the blastomere is detected from the enlarged specimen image. For edge detection, a known method such as a Sobel filter, a Laplacian filter, or a Canny filter can be used. Note that the above-described contour line extraction process when recognizing the first identifier 101 may be used, or a process of estimating and interpolating a portion where the contour line is interrupted may be additionally performed. After detecting the edge, a blast ball detection process is performed. As an example of the blast ball detection process, there is a method of detecting a circle using Hough transform. Alternatively, the blastomere may be detected using a known method such as ellipse fitting.

  FIG. 15B shows a portion detected by the blastomere detection process. For easier understanding, the portion detected as a blastomer is shown as a thick line in a form overlapping the enlarged specimen image. As shown in FIG. 15B, for example, a number or ID is temporarily attached to each detected blast ball in order to distinguish them. Here, blastomeres 1 to 4 are detected. Note that the number of blasts detected here can be registered as information 1007 indicating the second feature amount in the growth information database 144.

Next, a feature amount that is an index of uniformity of the blastomere is calculated. Here, the circularity and the diameter will be described. Here, the “circularity” is a value representing the circularity and can be defined by the following equation.
Circularity = 4πS ÷ L ²
S is the area (number of pixels) and L is the perimeter. This circularity indicates that when the value is 1, it is closest to a circle. In the example of FIG. 15B, the circularity can be calculated by using the number of pixels surrounded by the edge of each blast ball as S and using the peripheral length of the edge of each blast ball as L. “Diameter” is the diameter when the blastomer is approximately a circle, the maximum diameter when it is an ellipse, and the maximum length that passes through the center of gravity in other shapes such as a gourd.

  FIG. 15C shows an example in which the circularity and diameter are calculated for each blastomere. In this way, the feature amount may be calculated for each blast ball, and all these feature amounts may be registered as the growth information. From these plurality of feature amounts, a predetermined score for evaluating the growth stage is obtained. It may be calculated. Here, the uniformity (uniformity) score will be described.

As an example, a score indicating uniformity is defined as follows.
Uniformity = (size uniformity) + (deviation from circularity)
Here, the “size matching degree” is a value indicating the variation in the diameter of the blastomere, and the value becomes closer to 0 as the diameters are aligned, and the value increases as there is a variation. . For example, it can be defined by the following formula.
Size equality = variation coefficient = standard deviation ÷ average value In the example of FIG. 15C, the size uniformity is 0.365.

The “degree of deviation from circularity” is a value that considers deviation from the degree of circularity for all blastomeres. The value is close to 0 if each of the spheres is close to a circle, and the value is such that the deviation from the circle. Will grow. For example, it can be defined by the following formula.
Degree of deviation from circularity = 1-average value of circularity In the example of FIG. 15C, the degree of deviation from circularity is 0.25. Finally, the uniformity is “7.9” from the sum of the degree of size uniformity (0.365) and the degree of deviation from the circularity (0.25). In this way, scores calculated from a plurality of feature amounts may be registered in the growth information database 144. The score calculation method described here is an example, and other calculation methods may be used. For example, an important feature amount may be weighted. Moreover, when calculating a score, you may use the feature-value other than having demonstrated here.

  Next, a process for calculating the fragmentation ratio will be described. FIG. 16 shows an example of calculating the fragmentation ratio from the enlarged specimen image. In FIG. 16, the enlarged specimen image is shown by a simple illustration. First, an embryo area is extracted from the enlarged specimen image. Similar to the processing in FIG. 15, edge detection can be used to extract an embryo region. For edge detection, a known method such as a Sobel filter, a Laplacian filter, or a Canny filter can be used. Note that the above-described contour line extraction process when recognizing the first identifier 101 may be used, or a process of estimating and interpolating a portion where the contour line is interrupted may be additionally performed. After the edge detection, an embryo region detection process is performed. As an example of detection processing of an embryo region, there is a method of detecting a circle using Hough transform. Moreover, you may use well-known methods, such as ellipse fitting. FIG. 16B shows a portion detected by the embryo region detection process. For easier understanding, a portion detected as an embryo region is shown as a dotted line in a form overlapping the enlarged specimen image.

  Next, a fragmentation region is extracted. Fragmentation is cell debris (cell waste smaller in size than the blastomere) that is different from the blastomere in the embryo. As shown in the image of FIG. 16A, since the fragmentation region is a region where cell debris is gathered, the fragmentation region is extracted using a region division method. As a method of area division, known methods such as WaterShed, Split and Merge, Mean Shift, and Grabcut can be used. FIG. 16C shows an example in which a fragmentation region is extracted. Finally, the ratio of the fragmentation area to the portion detected as the embryo area is calculated. This ratio can be calculated from the number of pixels corresponding to each portion. In the example of FIG. 16C, the fragmentation ratio is 42%. The feature amount calculated in this way may be registered in the growth information database 144.

  Next, morphological evaluation performed at the time of the 4th to 5th days after the start of culture will be described. There is a method for evaluating the blastocyst grade 4 to 5 days after fertilization, and the Gardner classification is known. A blastocyst is a structure formed in the early stage of embryogenesis after formation of the cleavage space and before implantation. A blastocyst is composed of an inner cell mass and trophectoderm. The inner cell mass is a cell population formed inside a blastocyst and a cell population that will form a fetus in the future. The trophectoderm is a cell population in which the inner cell mass has different properties and will form part of the placenta in the future.

  According to Gardner's classification, the blastocyst development stage is evaluated in 6 stages, the inner cell mass and the trophectoderm are evaluated in 3 stages, and each combination is evaluated. As an example, FIG. 17 shows a stage of a blastocyst, and briefly shows the inner cell mass and trophectoderm. FIG. 18 is an example of evaluation of each of the inner cell mass and the trophectoderm. For example, the size of the inner cell mass and the homogeneity of trophoblasts are used as indicators.

  First, the evaluation of the inner cell mass will be described. The evaluation of the inner cell mass includes the size of the inner cell mass, the uniformity of the cells of the inner cell mass, whether the cells of the inner cell mass are in close contact with each other, or whether the number of cells in the inner cell mass is large. The feature amount calculation processing unit 153 calculates various feature amounts of the internal cell mass from the image information of the enlarged specimen image in the following manner. In FIG. 17, the region of the inner cell mass is clearly indicated by the outline, but actually, the inner cell mass is blurred in the enlarged specimen image inside the blastocyst. Therefore, as an example, the blastocyst is finely divided (in units of pixels or in units of several pixels), and it is determined whether each area is an area of an inner cell mass. The area of the inner cell mass can be defined by pixel value information, granularity (variation of pixel values), spatial frequency, or a combination of these information.

  The size of the inner cell mass is determined by the maximum length in the detected area (the maximum length passing through the center of gravity in the area) and the area of the area. The uniformity of the cells of the inner cell mass is determined by the granularity (pixel value variation) in the detected area. Similarly to the blastomere detection process described above, the number of cells in the inner cell mass may be calculated by detecting an edge in the area of the inner cell mass. Furthermore, it may be determined whether the cells of the inner cell mass are in close contact with each other based on the distance between the cells of the inner cell mass in the area (the distance between the center of gravity of each cell).

  The evaluation of the trophectoderm includes the number of cells in the trophectoderm and the uniformity in the trophectoderm. The feature amount calculation processing unit 153 calculates various feature amounts of the nutrient ectoderm from the image information of the enlarged specimen image in the following manner. First, a region other than the area of the inner cell mass described above is extracted as a region including the nutrient ectoderm. In the extracted area, edge detection processing and cell detection processing (Hough transform, elliptical fitting) are executed in the same manner as described above, and the number of cells in the nutrient ectoderm is calculated. The uniformity within the nutrient ectoderm can be determined by the variation in granularity of each cell detected by the above-described treatment. As another method, the homogeneity in the trophectoderm may be determined by counting the number of cells having a predetermined diameter or less among the cells detected by the above treatment.

  Although the morphological evaluation performed 2-3 days after fertilization and 4-5 days after fertilization was described above, morphological evaluation may be performed in the early stage of fertilization (after about 1 day). For example, morphological evaluation of pronuclear embryos may be used as an initial evaluation method. The pronuclear stage embryo is an embryo that has passed about ten or more hours after the spermatozoon. When the sperm enters the egg, the pronucleus derived from the ovum and the pronucleus derived from the sperm line up side by side. FIG. 19 shows the morphology of the two pronuclei and the nucleolus within the pronucleus. As shown in FIGS. 19A to 19C, there are differences in the size of the pronucleus, the distance between the two pronuclei, the arrangement of nucleoli in the pronucleus, and the like. In fertility treatment, it is known that there are differences in subsequent embryogenesis in terms of the size and arrangement of the pronucleus, and the size, arrangement, and symmetry of the nucleolus in the pronucleus.

  The feature amount calculation processing unit 153 may calculate various feature amounts of the pronuclear stage embryo from the image information of the enlarged specimen image in the following manner. Similarly to the above, edge detection processing and cell detection processing (Hough transform, elliptical fitting) are executed, and cells within a predetermined diameter range are determined as pronuclei. And for the part determined to be the pronucleus, the size of the pronucleus (diameter and area), the brightness of the pronucleus (brightness), the difference in size between the two pronuclei (diameter and area difference), The distance between the two anterior nuclei (the distance between the centers of gravity) is calculated.

  Further, in the same manner, the nucleolus detection process may be executed for the area determined to be the anterior nucleus. Cell detection processing (Hough transform, ellipse fitting) is executed, and cells in a predetermined diameter range (that is, a diameter smaller than a predetermined threshold) are determined as nucleolus. Then, the size (diameter and area) of the nucleolus, the number of nucleolus, the distance between the nucleolus, and the like are calculated. The feature amount calculated in this way may be registered in the growth information database 144.

  The feature amount calculated by the feature amount calculation processing unit 153 has been described above, but is not limited to the above. In the examples described above, evaluation of Veek, Gardner, and pronuclear embryos has been described, but some of these elements may be implemented, or other classifications similar to these may be used. In addition, cell features include the number of cells, cell shape, cell diameter, maximum length through the center of gravity of the cell area, circularity, cell aspect ratio, cell perimeter, cell area, cell Density (brightness and brightness), cell uniformity (size shift, pixel value variation, etc.), percentage of a given cell to the whole (fragmentation percentage, etc.), cell array (distance between cells and multiple And the combination of these).

  Here, the “number of cells” means the number of cells included in a predetermined region. The predetermined area may be an area of one cell or an area obtained by a plurality of cell populations such as an inner cell mass. Examples include the number of blastomeres contained in one fertilized egg and the number of cells contained in the inner cell mass. In the case of the pronuclear stage embryo, the “number of cells” may be the number of nucleolus in each of the two pronuclei contained in the fertilized egg, or the entire nucleus in the two pronuclei. It may be the number of small bodies. The “cell shape” means the shape of the contour line of at least one cell region, and may be the shape of one cell contour line, or the contour of a region obtained by a plurality of cell populations such as internal cell masses. It may be a line shape. The “cell diameter” means the diameter of at least one cell region, and is used when the shape of the cell is circular. The “cell diameter” may be the diameter of one cell region or the diameter of a region obtained by a plurality of cell populations such as an inner cell mass. The “maximum length that passes through the center of gravity of the cell region” means the maximum length that passes through the center of gravity of at least one cell region, and is used, for example, when the shape of the cell is other than a circle. The “maximum length that passes through the center of gravity of the cell region” may be the maximum length that passes through the center of gravity of one cell region, or the maximum length that passes through the center of gravity of a region obtained by a plurality of cell populations such as inner cell masses. It's okay. “Circularity” means the circularity described above. The “cell aspect ratio” means the ratio of the major axis (or long side) to the minor axis (or short side) of at least one cell region, and may be the ratio of the major axis to the minor axis of one cell. However, the ratio of the major axis to the minor axis of a region obtained by a plurality of cell populations such as an inner cell mass may be used. The “perimeter of the cell” means the perimeter of the outline of at least one cell region, and may be the perimeter of the outline of a single cell, or a plurality of cell populations such as an inner cell mass. The perimeter of the region obtained by The “cell area” means an area surrounded by an outline of at least one cell region, and may be the area of one cell or a region obtained by a plurality of cell populations such as an inner cell mass. May be the area. The “cell concentration” means the brightness or brightness of at least one cell, and may be the brightness or brightness of a single cell, or the brightness of a region obtained by a plurality of cell populations such as an inner cell mass. Or brightness. When a plurality of pixels are included, a predetermined value (average value, maximum value, minimum value, etc.) obtained from the plurality of pixels may be used as the “cell density”. “Cell uniformity” means a shift in cell size (diameter, area, etc.) and a variation in pixel values among a plurality of cells. In addition, “a ratio of a predetermined cell to the whole” means a ratio of at least one cell to an entire region. For example, it is the ratio of fragmentation (multiple cell debris) to the entire embryo area. The “cell arrangement” means an arrangement of a plurality of cells, and includes a distance between two cells and a positional relationship between the plurality of cells. In addition, the “cell arrangement” includes an arrangement of a plurality of cells included in one specific region (for example, a distance or positional relationship between a plurality of cells in the inner cell mass), a plurality of specific regions, and the like. The arrangement of a plurality of cells included (for example, the distance and positional relationship between nucleoli in each of the two pronuclei, and the symmetry of the arrangement of nucleoli in the two pronuclei) are also included.

  Further, the feature amount calculation processing unit 153 may determine the growth stage and pass / fail of the fertilized egg based on the feature amount described above. The feature amount calculation processing unit 153 may refer to the condition 1103 of the determination profile table 145 to determine whether the above-described feature amount satisfies a predetermined condition, and thereby output a grade 1102 that satisfies the condition. The grade 1102 information output here may be registered in the growth information database 144.

  Furthermore, the feature amount calculation processing unit 153 may switch the profile for determining the growth stage of the fertilized egg based on the elapsed time from the culture start date (reference time). FIG. 20 is a diagram for explaining a profile to be applied according to the elapsed time from the culture start date. As shown in FIG. 20, on the first day, the pronuclear embryo evaluation described above is used, on the second to third days, the Veek evaluation is used, and on the fourth to fifth days, the Gardner evaluation is used. You may do it. Since the determination profile table 145 also stores information on the application time (elapsed time from the reference time) 1101, the feature amount calculation processing unit 153 determines the reference time (for example, treatment date and time) and the acquisition time of the enlarged specimen image. By calculating the elapsed time from the above and referring to the determination profile table 145, a profile corresponding to the elapsed time can be applied. Here, the description has been given of switching the profile with the elapsed time from the reference time, but the present invention is not limited to this method. The feature amount calculation processing unit 153 may switch the profile based on the value of the specific feature amount after calculating the feature amount of the cell in the enlarged specimen image. In this case, an item representing a specific feature amount condition may be added to the determination profile table 145. Further, the feature amount calculation processing unit 153 may switch the profile based on the grade of the growth stage. For example, when a certain level of Veek has been reached at the time of the previous image acquisition, a method of switching to a Gardner evaluation profile may be used this time. This is made possible by holding grade information in the growth information database 144.

  The database creation processing unit 154 grows the well ID output from the first identifier recognition processing unit 151, the cell culture container ID output from the second identifier recognition processing unit 152, and the image information of the enlarged specimen image in association with each other. Store in the information database 144. Furthermore, the database creation processing unit 154 includes information on the presence / absence of a fertilized egg determined by the feature amount calculation processing unit 153, information on various feature amounts calculated by the feature amount calculation processing unit 153, and the acquisition time of the enlarged specimen image. Information is stored in the growth information database 144 in association with the cell culture container ID and the well ID.

  Next, the flow of processing in the growth information management apparatus 130 will be described. FIG. 21 is a flowchart for explaining the flow of processing in the growth information management system 1.

  First, a growth information management program is activated on a predetermined information terminal (2101). Next, the second identifier 102 of the cell culture container 100 is read by the second identifier reader 120 (2102). Next, the second identifier recognition processing unit 152 refers to the second identifier correspondence table 143 using the read information of the second identifier 102. Then, the second identifier recognition processing unit 152 outputs the cell culture container ID corresponding to the second identifier 102, and temporarily records information on the cell culture container ID in a storage device or the like (2103). Next, the 2nd identifier recognition process part 152 determines the alignment type of the 1st identifier 101 for every cell culture container from the information of the container type 903 of the 2nd identifier corresponding | compatible table 143 (2104). This first identifier information is output to the first identifier recognition processing unit 151, whereby the first identifier correspondence table corresponding to the container type 903 is selected in the subsequent processing.

  Next, the operator operates the image acquisition apparatus 110 using the input unit 132 to adjust the focus so that the microwell 105 and the first identifier 101 are included in the frame (2105). Next, the image acquisition apparatus 110 is operated to acquire an enlarged specimen image including the microwell 105 and the first identifier 101 (2106). Next, the first identifier recognition processing unit 151 determines whether the microwell 105 and the first identifier 101 are present in the enlarged specimen image (2107). This can be determined by detecting the contour line of the microwell 105 and the contour line of the first identifier 101 after the contour line extraction image is created from the enlarged specimen image. Here, if the microwell 105 and the first identifier 101 are not present in the enlarged specimen image, the output unit 133 warns that effect (2108), and returns to step 2105.

  Next, time information when the enlarged specimen image is acquired is acquired (2109). Next, the first identifier recognition processing unit 151 recognizes the first identifier 101 in the enlarged specimen image, and outputs the corresponding well ID by referring to the first identifier correspondence table (2110). Next, the first identifier recognition processing unit 151 displays the cell culture container ID and the well ID on the output unit 133 and inquires of the operator whether these pieces of information are correct (2111). If it is wrong, the process returns to Step 2102.

  Next, the feature amount calculation processing unit 153 calculates the feature amount of the cell from the enlarged specimen image (2112). The processing flow here will be described later. Thereafter, the database creation processing unit 154 associates the well ID output from the first identifier recognition processing unit 151, the cell culture container ID output from the second identifier recognition processing unit 152, and the image information of the enlarged specimen image. Registered in the growth information database 144 (2113). Further, the database creation processing unit 154 displays information on the presence / absence of a fertilized egg determined by the feature amount calculation processing unit 153, information on various feature amounts, and information on the acquisition time of the enlarged specimen image, the cell culture container ID and the well ID. And is registered in the growth information database 144 (2114).

  FIG. 22 is a flowchart for explaining a specific processing flow of step 2112 in FIG. The subject of the following processing is the feature amount calculation processing unit 153. First, the presence or absence of a fertilized egg is determined from the enlarged specimen image (2201). Next, the profile to be used is selected by referring to the determination profile table 145 (2202). For example, by calculating the elapsed time from the treatment date and time and the acquisition time of the enlarged specimen image and referring to the application time 1101 of the determination profile table 145, a profile corresponding to the elapsed time can be selected.

  Next, a feature amount corresponding to the selected profile is calculated (2203). For example, in the case of the first record in FIG. 11, the feature amount of the blast ball and fragmentation is calculated as the feature amount. Next, a score is calculated based on the calculated feature amount (2204). Finally, the grade is determined by referring to the determination profile table 145 (2205). Here, it is determined whether or not the feature amount calculated in step 2203 satisfies the condition 1103 of the determination profile table 145. If the condition is satisfied, the corresponding grade 1102 is output. The score and grade information may then be displayed on the output unit 133 or registered in the growth information database 144.

  As described above, according to the present embodiment, since the well ID is automatically associated with the enlarged specimen image, there is no need to manually associate the enlarged specimen image with the growth information. The risk of misplacement is reduced. Further, not only the well ID but also the cell culture container ID can be associated with the enlarged specimen image, and all the specimen information can be uniquely identified for the enlarged specimen image. Further, since not only the feature quantity but also the information about the image acquisition time is associated with the enlarged specimen image, it becomes easy to manage information relating to the determination of the growth stage of the fertilized egg. In addition, conventionally, an operator who has viewed an enlarged specimen image visually determines the feature amount of a cell and determines the growth stage of a fertilized egg. The feature amount is calculated, and the growth stage of the fertilized egg can be automatically determined based on the feature amount.

  Next, another embodiment of the present invention will be described. FIG. 23 is a diagram showing another form of the growth information database 144. The same components as those in FIG. 10 are denoted by the same reference numerals and description thereof is omitted. From the image acquisition device 110 such as a microscope, it is also possible to acquire depth information when an image is acquired. Therefore, an item of depth information (depth from the uppermost end of the fertilized egg) 1009 may be added to the growth information database 144, and the depth information 1009 may be managed together with the growth information 1003.

  By managing the depth information 1009, not only the two-dimensional image information 1004 but also three-dimensional image information can be managed. For example, the image information 1004 is acquired at a plurality of depths for a certain cell (for example, the first to third records in FIG. 23). The growth information management system 1 may create a three-dimensional polygon graphic by laminating a plurality of images corresponding to the plurality of depths (superimposing them in the depth direction). For modeling from a two-dimensional image to a three-dimensional polygon, a method well known to those skilled in the art may be used. Thereby, the three-dimensional information of a fertilized egg can also be managed. Further, by referring to the three-dimensional polygon, it is possible to determine the development stage of the fertilized egg by a three-dimensional display. In this embodiment, the cell volume may be calculated using the information of the three-dimensional polygon, and the cell volume may be registered in the growth information database 144 as a feature amount. Here, the “cell volume” means the volume of at least one cell. The “cell volume” may be the volume of the whole fertilized egg or the volume of a specific region (at least one cell) in the fertilized egg. Further, the shape of the entire fertilized egg may be determined from the information of the three-dimensional polygon, and the shape information may be registered in the growth information database 144 as a feature amount.

  In addition, this invention is not limited to embodiment mentioned above, Other various modifications are included. For example, the above-described embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to the one having all the configurations described. In addition, a part of the configuration of an embodiment may be replaced with the configuration of another embodiment, and the configuration of another embodiment may be added to the configuration of an embodiment. In addition, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

  For example, as described above, the constituent elements of the growth information management device 130 may be distributed to a plurality of information processing devices on the network. For example, the growth information management system may include a terminal (first information processing device) connected to the image acquisition device 110 and a server (second information processing device) including a storage device. Various tables, databases, and the like are stored in the terminal or server according to the corresponding processing. As an example, steps 2101 to 2111 in FIG. 21 may be performed at the terminal, and steps 2112 to 2114 may be performed at the server. In this case, in step 2111, the terminal transmits various types of information (cell culture vessel ID, well ID, enlarged specimen image) to the server, and the server receives the various types of information, extracts feature values, and registers them in the storage device. Just do. In another example, the process of associating the well ID with the first identifier 101 may be performed on the server side. In this case, the terminal may transmit the information on the enlarged specimen image together with the information on the cell culture container ID to the server, and the server may execute subsequent processing.

  Although the example of a human fertilized egg was demonstrated as the culture | cultivation object above, it is not limited to this. Examples of cells to be cultured include fertilized eggs, egg cells, ES cells (embryonic stem cells), and iPS cells (artificial pluripotent stem cells). An egg cell refers to an unfertilized egg cell, and includes an immature oocyte and a mature oocyte. After fertilization, the fertilized egg increases in number of cells from the 2 cell stage, the 4 cell stage, and the 8 cell stage by cleavage, and develops into a blastocyst through a morula. Fertilized eggs include early embryos such as 2-cell embryos, 4-cell embryos and 8-cell embryos, morulas, blastocysts (including early blastocysts, expanded blastocysts and escaped blastocysts). A blastocyst means an embryo composed of external cells with the potential to form the placenta and internal cell masses with the potential to form embryos. ES cells refer to undifferentiated pluripotent or totipotent cells obtained from the inner cell mass of a blastocyst. An iPS cell refers to a cell having a pluripotency similar to that of an ES cell by introducing several types of genes (transcription factors) into somatic cells (mainly fibroblasts). That is, the cell includes an aggregate of a plurality of cells such as a fertilized egg and a blastocyst.

  Also, the present invention is preferably suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, eg, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and livestock such as cattle, horses and pigs. Is mentioned. The present invention is particularly suitable for culturing human fertilized eggs.

  Further, as described above, each processing unit of the growth information management device 130 may be realized by a program code of software that realizes the function of the embodiment. In this case, a storage medium in which the program code is recorded is provided to the information processing apparatus, and the information processing apparatus (or CPU) reads the program code stored in the storage medium. In this case, the program code itself read from the storage medium realizes the functions of the above-described embodiment, and the program code itself and the storage medium storing the program code constitute the present invention. As a storage medium for supplying such program code, for example, a flexible disk, CD-ROM, DVD-ROM, hard disk, optical disk, magneto-optical disk, CD-R, magnetic tape, nonvolatile memory card, ROM Etc. are used.

  In addition, the information lines in the drawings indicate what is considered necessary for the description, and do not necessarily indicate all information lines on the product. All the components may be connected to each other.

1: Growth information management system 100: Cell culture container 101: 1st identifier 102: 2nd identifier 103: Bottom part 104: Side wall 105: Microwell 106: Cell accommodating part 107: 3rd identifier 110: Image acquisition apparatus 120: 2nd Identifier reader 130: Specimen image management device 131: Storage device 132: Input unit 133: Output unit 141: Specimen information database 142: Well design information 143: Second identifier correspondence table 144: Growth information database 145: Determination profile table 151: First identifier recognition processing unit 152: Second identifier recognition processing unit 153: Feature amount calculation processing unit 154: Database creation processing unit

Claims (13)

A plurality of micro-wells, and cell culture vessel having a first identifier assigned become the microwells paired,
Storage means for storing at least the correspondence between the enlarged specimen image including the pair of the first identifier and the microwell, the information on the first identifier, and the information on the feature amount of the cell in the microwell. ,
First identifier recognition means for recognizing and acquiring the first identifier in the enlarged specimen image;
A feature amount calculating means for calculating a feature amount of a cell in the enlarged specimen image;
Registration means for registering in the storage means at least correlated with the information on the feature amount and the magnified specimen image and the first identifier,
A growth information management system.   The feature amount includes the number of cells, cell shape, cell diameter, maximum length passing through the center of gravity of the cell area, circularity, cell aspect ratio, cell perimeter, cell area, cell concentration, cell The growth information management system according to claim 1, comprising at least one of: uniformity of a cell, a ratio of a predetermined cell to a whole cell, a cell arrangement, and a cell volume.   The feature quantity calculating means calculates a plurality of feature quantities of different cells in the enlarged specimen image, and calculates a predetermined score for evaluating a growth stage based on the plurality of feature quantities. The growth information management system according to 2.   The feature amount calculation means determines whether the feature amount satisfies a predetermined condition using determination information for determining the growth stage of the cell, and determines the growth stage of the cell. The growth information management system according to any one of the above.   The feature amount calculation means switches the determination information for determining the growth stage of the cell based on the elapsed time from the start of culturing the cell, the feature amount, or the growth stage of the cell. 4. The growth information management system according to 4. The registration unit associates depth information related to the cell depth when the enlarged specimen image is acquired with the storage means in association with the enlarged specimen image, information about the first identifier, and information about the feature amount. The growth information management system according to any one of claims 1 to 5, which is registered. A plurality of micro-wells, said growth information management processing for cell culture vessel having a first identifier assigned become microwells paired, to be executed by the at least comprising the information processing apparatus calculating means and storage means A program,
In the calculation means,
A first identifier recognition process for recognizing and acquiring the first identifier in an enlarged specimen image including a pair of the first identifier and the microwell;
A feature amount calculation process for calculating a feature amount of a cell in the enlarged specimen image;
A registration process for registering in the storage means and information on the feature amount and the magnified specimen image and the first identifier at least association,
A program for running   The feature amount includes the number of cells, cell shape, cell diameter, maximum length passing through the center of gravity of the cell area, circularity, cell aspect ratio, cell perimeter, cell area, cell concentration, cell The program according to claim 7, comprising at least one of: uniformity of a cell, a ratio of a predetermined cell to a whole cell, a cell arrangement, and a cell volume.   The feature quantity calculation processing includes calculating a plurality of feature quantities of different cells in the enlarged specimen image, and calculating a predetermined score for evaluating a growth stage based on the plurality of feature quantities. Item 9. The program according to item 7 or 8.   The feature amount calculation process includes determining whether the feature amount satisfies a predetermined condition using determination information for determining the growth stage of the cell, and determining the growth stage of the cell. The program according to any one of 7 to 9.   The feature amount calculation processing includes switching the determination information for determining the growth stage of the cell based on the elapsed time from the start of culturing the cell, the feature amount, or the growth stage of the cell. The program according to claim 10. In the registration process, the depth information related to the cell depth when the enlarged specimen image is acquired is associated with the enlarged specimen image, the information about the first identifier, and the information about the feature amount in the storage unit. The program according to any one of claims 7 to 11, including registration. A plurality of micro-wells, the cell culture vessel having a first identifier assigned become the microwells paired image acquisition means for acquiring a magnified specimen image including a pair of the said first identifier microwell A first information processing apparatus comprising:
It said enlarged specimen image, and the first identifier, and the information on the characteristics of cells in the microwells, and a second information processing apparatus including a storage means for storing at least association with a
With
The first information processing apparatus recognizes and acquires the first identifier in the enlarged specimen image, transmits information on the enlarged specimen image and the first identifier to the second information processing apparatus,
The second information processing apparatus receives information on the enlarged specimen image and the first identifier from the first information processing apparatus, calculates a feature amount of a cell in the enlarged specimen image, and the enlarged specimen image A growth information management system that registers at least the information about the first identifier and the information about the feature amount in association with each other.

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