WO2019163369A1

WO2019163369A1 - Determination apparatus, cell detachment/collection apparatus, and determination method

Info

Publication number: WO2019163369A1
Application number: PCT/JP2019/001886
Authority: WO
Inventors: 慎市菊池; 貴之占部
Original assignee: 富士フイルム株式会社
Priority date: 2018-02-20
Filing date: 2019-01-22
Publication date: 2019-08-29
Also published as: JP7030953B2; JPWO2019163369A1

Abstract

A determination apparatus is provided with: a pattern-drawn surface having a pattern drawn thereon; an imaging unit of which the imaging field of view is preset to the pattern-drawn surface; and a determination unit whereby it becomes possible to determine the presence or absence of cells attached to a light-permeable culturing surface of a culture vessel for cell culturing use, wherein the determination is carried out based on an image of the pattern which is obtained by the imaging unit while placing the culturing surface between the pattern-drawn surface and the imaging unit.

Description

Determination device, cell detachment recovery device, and determination method

The present invention relates to a determination apparatus, a cell detachment collection apparatus, and a determination method.

The following technologies are known as cell culture device technologies. For example, Japanese Patent Application Laid-Open No. 2014-113062 discloses a scraper for scraping off cells adhering to the bottom surface of a culture container, a pipette device for sucking cells detached from the bottom surface of the culture container together with a medium, and a treatment with the pipette device. A cell detachment system is described, which includes a cell observation device that images the obtained culture vessel and determines whether or not cells remain in the culture vessel.

The subculture treatment in the adhesion culture in which the cells are adhered to the culture surface of the culture vessel is cultured, and the cells adhered to the culture surface of the culture vessel are separated from the culture surface using a chemical solution such as an enzyme agent, In this process, detached cells are collected, and the collected cells are cultured in a new culture vessel. In the passaging treatment, if there are many cells remaining on the culture surface after the detachment treatment, cell loss occurs and the culture efficiency is lowered. Therefore, the presence or absence of cells remaining on the culture surface after the detachment treatment is determined, and the detachment treatment is continued or restarted when the amount of remaining cells is a certain level or more. However, when the presence / absence of cells remaining on the culture surface is visually determined by an operator, the accuracy of determining the presence / absence of cells varies among workers, and as a result, the amount of collected cells varies among workers. There is a problem.

Patent Document 1 describes that the presence or absence of cells remaining in the culture container is determined from an image obtained by imaging the culture container after the peeling treatment. However, Patent Document 1 does not describe a specific method for determining the presence or absence of cells remaining in the culture vessel. For example, if the cells are light transmissive, it is difficult to specify the area where the cells remain from the image obtained by imaging the culture container. Therefore, it is difficult to determine the presence or absence of cells remaining in the culture container. It is considered difficult.

The disclosed technique has been made in view of the above points, and aims to accurately determine the presence or absence of cells attached to the culture surface of a culture vessel.

The determination apparatus according to the disclosed technique includes imaging a drawing surface on which a pattern is drawn, an imaging unit in which an imaging field of view is set on the drawing surface, and a culture surface having a light transmission property of a culture vessel for culturing cells. And a determination unit that determines the presence / absence of cells attached to the culture surface based on the image of the pattern acquired by the imaging unit in a state of being disposed between the two.

According to the determination apparatus according to the disclosed technology, it is possible to accurately determine the presence / absence of cells attached to the culture surface of the culture container even when the cells have light permeability.

The determination unit may determine the presence or absence of cells attached to the culture surface based on at least one of distortion and blur in the image portion corresponding to the contour line of the pattern. This embodiment is particularly effective for cells having light permeability.

The pattern may include a bright part and a dark part arranged adjacent to each other. As a result, the determination in the determination unit can be performed more accurately.

The pattern may have a repeating pattern in which bright parts and dark parts are alternately arranged. Thereby, the determination process in the determination unit can be simplified, and the processing time can be shortened.

The determination apparatus according to the disclosed technique may further include a derivation unit that derives the area of the region where the cells are attached to the culture surface. In addition, the determination apparatus according to the disclosed technique may further include a derivation unit that derives a ratio of the area of the region where the cells are attached to the culture surface to the area of the culture surface. As a result, the determination unit can perform more accurate determination in consideration of the cell recovery efficiency and the processing cost.

A part of the pattern may have light transparency, and in this case, the determination device according to the disclosed technique further includes a light source disposed on the opposite side of the imaging unit with the drawing surface interposed therebetween. Also good. The pattern may be formed of a light emitting element. According to these aspects, the contrast of the pattern can be increased, and the determination accuracy in the determination unit can be increased.

The drawing surface may include a plurality of pixels arranged in a matrix, and a pattern may be formed by controlling a light transmission state or a light emission state of each of the plurality of pixels. According to this aspect, it is possible to appropriately change the pattern of the design.

The imaging unit may acquire a picture image in a state where the entire culture surface is included in the imaging field of view. According to this aspect, when it is determined whether or not the cells are attached to the entire culture surface, the number of times of imaging by the imaging unit can be reduced.

A cell detachment collection apparatus according to the disclosed technology includes the above-described determination apparatus, a thermostatic bath that has a storage space for storing the culture container, keeps the temperature of the storage space constant, a transport unit that transports the culture container, and a culture A chemical solution adding unit for adding a chemical solution for separating cells attached to the surface to the culture vessel; a chemical solution removing unit for removing the chemical solution from the culture vessel; and a cell recovery unit for collecting the cells detached from the culture surface.

According to the peeling recovery apparatus according to the disclosed technique, the same effect as that of the determination apparatus according to the disclosed technique can be obtained, and further, a peeling process for peeling cells adhering to the culture surface, and adhesion to the cell surface It is possible to automate the determination process for determining the presence of cells.

In the determination method according to the disclosed technique, the imaging field of the imaging unit is set on the drawing surface on which the pattern is drawn, and the culture surface having light permeability of the culture vessel for culturing cells between the drawing surface and the imaging unit The image of the pattern is acquired by the imaging unit in a state where the is placed, and the presence or absence of cells attached to the culture surface is determined based on the image of the pattern.

According to the determination method according to the disclosed technique, it is possible to accurately determine the presence or absence of cells attached to the culture surface of the culture vessel even when the cells have light permeability.

Determine the presence or absence of cells adhering to the culture surface of the culture vessel.

It is a figure which shows an example of a structure of the determination apparatus which concerns on embodiment of the technique of an indication. It is a perspective view which shows an example of a structure of the culture container which concerns on embodiment of the technique of an indication. FIG. 2B is a cross-sectional view taken along line 2B-2B in FIG. 2A. It is a figure which shows an example of the pattern drawn on the drawing surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the pattern drawn on the drawing surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the pattern drawn on the drawing surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the hardware constitutions of the determination part which concerns on embodiment of the technique of an indication. 14 is a flowchart illustrating an example of a flow of determination processing in a determination unit according to an embodiment of the disclosed technology. It is a figure showing an example of composition of a cell exfoliation collection device concerning an embodiment of an art of an indication provided with a judgment device concerning an embodiment of an art of an indication. 10 is a flowchart illustrating an example of an operation sequence of the cell detachment collection apparatus according to an embodiment of the disclosed technology. It is a figure which shows an example of the image of the pattern acquired by the imaging part which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the image of the pattern acquired by the imaging part which concerns on embodiment of the technique of an indication. It is a figure which shows a mode that the cell is collect | recovered in the cell collection part which concerns on embodiment of the technique of an indication. It is a figure which shows a mode that the cell is collect | recovered in the cell collection part which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the other method of positioning the culture container which concerns on embodiment of the technique of an indication to an imaging position. It is a figure which shows an example of the other method of positioning the culture container which concerns on embodiment of the technique of an indication to an imaging position. It is a figure which shows the other example of a structure of the culture container which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the other method of removing an enzyme agent from the culture surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the other method of removing an enzyme agent from the culture surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the other method of removing an enzyme agent from the culture surface which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the other method of collect | recovering the cells which peeled from the culture surface of the culture container which concerns on embodiment of the technique of an indication. It is a figure which shows the other example of a structure of the determination apparatus which concerns on embodiment of the technique of an indication. It is a figure which shows the other example of a structure of the determination apparatus which concerns on embodiment of the technique of an indication. It is a figure which shows the other example of a structure of the determination apparatus which concerns on embodiment of the technique of an indication. 14 is a flowchart illustrating an example of a flow of derivation processing in a derivation unit according to an embodiment of the disclosed technology. 14 is a flowchart illustrating another example of the flow of derivation processing in the derivation unit according to an embodiment of the disclosed technology. It is a figure which shows the other example of the determination method using the determination apparatus which concerns on embodiment of the technique of an indication. It is a figure which shows an example of the image of the pattern imaged through the culture surface of the state which has not adhered the cell based on embodiment of the technique of an indication. It is a figure which shows an example of the image of the pattern imaged through the culture surface of the state which the cell has adhered based on embodiment of the technique of an indication.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, substantially the same or equivalent components or parts are denoted by the same reference numerals.

FIG. 1 is a diagram illustrating an example of a configuration of a determination apparatus 1 according to an embodiment of the disclosed technology. The determination apparatus 1 includes a drawing surface 20 on which a pattern 21 is drawn, an imaging unit 30 in which an imaging field of view is set on the drawing surface 20, and a determination unit 40. The determination unit 40 adheres to the culture surface 11 based on the image of the pattern 21 acquired by the imaging unit 30 in a state where the culture surface 11 of the culture vessel 10 is disposed between the drawing surface 20 and the imaging unit 30. Determine the presence or absence of cells.

2A is a perspective view showing an example of the configuration of the culture vessel 10, and FIG. 2B is a cross-sectional view taken along line 2B-2B in FIG. 2A. Although the external shape of the culture vessel 10 is not particularly limited, for example, it may be a rectangular parallelepiped including a top plate 10A and a bottom plate 10B facing the top plate 10A as shown in FIG. 2A. The top plate 10A of the culture vessel 10 is provided with two caps 12 that can be opened and closed. By opening the cap 12, it is possible to inject a chemical solution such as a medium and an enzyme into the culture vessel 10, discharge the chemical solution from the culture vessel 10, and take out the cells from the culture vessel 10. . By closing the cap 12, the internal space of the culture vessel 10 is sealed.

The inner surface of the culture vessel 10 of the bottom plate 10 B is a culture surface 11, and the cells 200 cultured in the culture vessel 10 are cultured while being adhered to the culture surface 11. In the culture container 10, at least the top plate 10A and the bottom plate 10B are light transmissive. When determining the presence or absence of cells adhering to the culture surface 11 using the determination device 1, the culture container 10 is disposed between the drawing surface 20 and the imaging unit 30. At this time, the culture container 10 is arranged in such a direction that the bottom plate 10B is on the drawing surface 20 side and the top plate 10A is on the imaging unit 30 side. Note that the culture vessel 10 may be arranged in such a direction that the bottom plate 10B is on the imaging unit 30 side and the top plate 10A is on the drawing surface 20 side.

The drawing surface 20 may be constituted by, for example, the surface of a plate-like member, a sheet-like member, or a film-like member, and the pattern 21 may be drawn on the surface of these members. The area of the region where the pattern 21 is drawn on the drawing surface 20 is preferably larger than the area of the culture surface 11 of the culture vessel 10.

FIG. 3A, FIG. 3B, and FIG. 3C are diagrams showing an example of the picture 21 drawn on the drawing surface 20, respectively. The pattern 21 includes a bright part 21A and a dark part 21B. In the pattern 21, it is preferable that the boundary between the bright part 21 A and the dark part 21 B extends over the entire drawing surface 20. Further, as shown in FIGS. 3A to 3C, the pattern 21 further preferably includes a repetitive pattern in which bright portions 21A and dark portions 21B are alternately arranged.

The pattern 21 may form a so-called check pattern in which square light portions 21A and dark portions 21B are alternately arranged in the vertical direction and the horizontal direction, as shown in FIG. 3A, for example. In addition, for example, as shown in FIG. 3B, the pattern 21 forms a so-called stripe pattern in which linear bright portions 21 A and dark portions 21 B extending in one direction are alternately arranged in the intersecting direction intersecting the one direction. You may do. Further, for example, as illustrated in FIG. 3C, the pattern 21 includes a plurality of linear dark portions 21 B extending in the first direction at a certain interval in the second direction intersecting the first direction. In addition, a plurality of linear dark portions 21B extending in the second direction are arranged at a certain interval in the first direction, and bright portions 21A are arranged in a region other than the dark portion 21B. A so-called lattice pattern may be formed.

When the drawing surface 20 is constituted by the surface of a member that does not transmit light, a portion of the drawing surface 20 drawn in white, for example, is a bright portion 21A, and a portion of the drawing surface 20 drawn in black, for example, is It is good also as the dark part 21B. On the other hand, when the drawing surface 20 is constituted by the surface of a light-transmitting member, for example, a portion where the light transmittance of the drawing surface 20 is reduced by coloring the drawing surface 20 is set as a dark portion 21B. A portion where the light transmittance of the drawing surface 20 is maintained by not coloring the surface 20 may be a bright portion 21A.

The imaging unit 30 includes an imaging device such as a digital camera, for example. The imaging unit 30 removes the pattern 21 drawn on the drawing surface 20 under the condition that the liquid such as the cell culture solution or the enzyme agent does not enter the photographing optical path through the top plate 10A and the bottom plate 10B of the culture vessel 10. Take an image. That is, the image of the pattern 21 drawn on the drawing surface 20 is formed on the imaging surface of the imaging unit 30 via the bottom plate 10B and the top plate 10A of the culture vessel 10. It is preferable that the entire culture surface 11 is included in the imaging field of the imaging unit 30. The imaging unit 30 generates image data of the pattern 21 and supplies the generated image data to the determination unit 40.

The determination unit 40 performs determination processing for determining the presence or absence of cells attached to the culture surface 11 by analyzing the image data of the pattern 21 generated by the imaging unit 30. Here, FIG. 4 is a diagram illustrating an example of a hardware configuration of the determination unit 40. The determination unit 40 includes a computer 500. That is, the determination unit 40 includes a communication interface (I / F) for performing communication with a CPU (Central Processing Unit) 501, a main storage device 502 as a temporary storage area, a nonvolatile auxiliary storage device 503, and the imaging unit 30. InterFace) 504 and a display unit 505 such as a liquid crystal display. The CPU 501, main storage device 502, auxiliary storage device 503, communication I / F 504, and display unit 505 are each connected to a bus 507. The auxiliary storage device 503 stores a determination program 506 describing the procedure of the determination process. The determination unit 40 performs determination processing when the CPU 501 executes the determination program 506.

FIG. 5 is a flowchart showing an example of the flow of determination processing in the determination unit 40. In step S 1, the determination unit 40 acquires the image data of the pattern 21 generated by the imaging unit 30.

In step S 2, the determination unit 40 analyzes the image data of the pattern 21, and determines whether distortion or blur has occurred in the image portion corresponding to the outline of the pattern 21. The outline of the pattern 21 means the boundary between the bright part 21A and the dark part 21B.

Here, it is assumed that the cells cultured in the culture vessel 10 are transparent or translucent to visible light. Therefore, when cells are attached to the culture surface 11 of the culture vessel 10, the image of the pattern 21 drawn on the drawing surface 20 is imaged on the imaging surface of the imaging unit 30 via the cells attached to the culture surface. The In this case, in the image of the pattern 21 acquired by the imaging unit 30, an image portion corresponding to the contour line of the pattern 21 (the boundary between the bright part 21A and the dark part 21B) has a refractive index between the cell and the medium surrounding the cell. Distortion or blur is caused by effects such as differences and scattering of light within the cell. The distortion in the contour line means that the boundary line between the bright part 21A and the dark part 21B is curved. The blurring of the outline means that the contrast of the bright part 21A and the dark part 21B is lowered. FIG. 19A is an example of an image of the picture 21 captured through the culture surface 11 with no cells attached. FIG. 19B is an example of an image of the pattern 21 captured through the culture surface 11 in a state where cells are attached. As apparent from comparison between FIG. 19A and FIG. 19B, when cells are attached to the culture surface 11, the image portion corresponding to the contour line of the picture 21 (the boundary between the bright part 21 A and the dark part 21 B) is distorted. It can be confirmed that blur is occurring.

When the image portion of the contour line of the pattern 21 is blurred, it can be considered that the spatial frequency is lowered in the image of the pattern 21. Therefore, the determination unit 40 uses, for example, the spatial frequency or contrast of the image of the pattern 21 acquired in advance in a state where no cells are attached to the culture surface 11 as a reference, and the spatial frequency or contrast of the image of the pattern 21 to be determined. When the difference from the reference is equal to or greater than the threshold value, it may be determined that the image portion corresponding to the contour line of the pattern 21 is blurred. In addition, the determination unit 40 uses, for example, the contour line in the image of the pattern 21 acquired in advance in a state where cells are not attached to the culture surface 11 as a reference, and the contour line in the image of the pattern 21 to be determined is based on the above reference. When the amount of deviation is greater than or equal to the threshold value, it may be determined that the image portion corresponding to the contour line of the pattern 21 is distorted.

If the determination unit 40 determines that the image portion corresponding to the contour line of the pattern 21 is distorted or blurred, it is determined in step S3 that “there are cells attached to the culture surface 11 of the culture vessel 10”. To do. On the other hand, when the determination unit 40 determines that the image portion corresponding to the contour line of the pattern 21 is not distorted or blurred, in step S4, “no cells attached to the culture surface 11 of the culture vessel 10”. Is determined.

In step S5, the determination unit 40 outputs a determination result. For example, the determination unit 40 may display the determination result on a display (not shown). Further, the determination unit 40 may output the determination result by voice, or may output it by text printed on paper.

FIG. 6 is a diagram illustrating an example of a configuration of the cell detachment / collection apparatus 2 according to an embodiment of the disclosed technology, which includes the determination apparatus 1. In addition to the drawing surface 20, the imaging unit 30, and the determination unit 40 that constitute the determination device 1, the cell detachment / collection device 2 includes a thermostatic bath 110, a transport unit 120, a cap opening / closing unit 130, a chemical solution removal unit 140, a chemical solution addition unit 150, and A cell recovery unit 160 is provided.

The thermostat 110 has a storage space for storing the plurality of culture vessels 10 and keeps the temperature of the storage space constant (for example, 37 ° C.).

The transport unit 120 is an arm type robot having a gripping mechanism 121 that grips the culture vessel 10. The transport unit 120 transports the culture container 10 between the thermostatic chamber 110, the cap opening / closing unit 130, the chemical solution removal unit 140, the chemical solution addition unit 150, and the cell recovery unit 160 in a state where the culture container 10 is gripped by the gripping mechanism 121. Do. The conveyance part 120 can change the attitude | position of the culture container 10 freely in the state which hold | gripped the culture container 10. FIG.

The cap opening / closing unit 130 includes a gripping mechanism 131 that grips the cap 12 provided in the culture vessel 10, and opens and closes the cap 12 by rotating the cap 12 while the cap 12 is gripped by the gripping mechanism 131. .

The chemical solution removal unit 140 includes a suction nozzle 141 and a waste bottle 142, and removes the chemical solution from the culture vessel 10 by sucking the chemical solution such as a medium and an enzyme contained in the culture vessel 10 from the suction nozzle 141. . The chemical liquid sucked from the suction nozzle 141 is collected in the waste bottle 142.

The chemical solution addition unit 150 has a discharge nozzle 151 and a chemical solution bottle 152 that individually stores chemical solutions such as a culture medium and an enzyme agent, and the chemical solution stored in the chemical solution bottle 152 is discharged from the discharge nozzle 151, whereby the culture container 10. Add the chemical inside. In addition, an enzyme agent is used in the peeling process which peels the cell adhering to the culture surface 11 of the culture container 10. FIG.

The cell collection unit 160 includes a suction nozzle 161 and a collection bottle 162, and sucks the cells detached from the culture surface 11 of the culture vessel 10 by the separation process from the suction nozzle 161 and collects them in the collection bottle 162.

Hereinafter, an example of the operation of the cell detachment collection apparatus 2 will be described with reference to FIG. FIG. 7 is a flowchart showing an example of an operation sequence of the cell detachment / collection apparatus 2.

In the initial state, it is assumed that the culture vessel 10 is accommodated in the thermostatic chamber 110, and the cells are cultured in a state where the cells adhere to the culture surface 11 in the culture vessel 10. Further, it is assumed that the culture medium is accommodated in the culture vessel 10 together with the cells.

In step S11, the transport unit 120 takes the culture vessel 10 out of the thermostatic chamber 110 and transports it to the cap opening / closing unit 130. In step S 12, the cap opening / closing part 130 opens the cap 12 of the culture vessel 10. In step S 13, the transport unit 120 transports the culture container 10 to the chemical solution removal unit 140. In step S 14, the chemical solution removing unit 140 inserts the suction nozzle 141 into the culture container 10 from the circulation port of the culture container 10 exposed by opening the cap 12, and sucks the medium contained in the culture container 10 as the suction nozzle. The medium is removed from the culture vessel 10 by aspiration from 141.

In step S15, the transport unit 120 transports the culture vessel 10 to the chemical solution addition unit 150. In step S 16, the chemical solution addition unit 150 inserts the discharge nozzle 151 into the culture container 10 from the circulation port of the culture container 10 exposed by opening the cap 12, and discharges the enzyme agent from the discharge nozzle 151. An enzyme agent is added to the culture vessel 10. In step S 17, the transport unit 120 transports the culture container 10 to the cap opening / closing unit 130. In step S18, the cap opening / closing part 130 closes the cap 12 of the culture vessel 10. In step S 19, the transport unit 120 stores the culture container 10 in the thermostatic chamber 110. While the culture container 10 is accommodated in the thermostatic chamber 110, the separation of the cells from the culture surface 11 proceeds.

In step S20, the transport unit 120 determines whether or not a predetermined time has elapsed since the culture vessel 10 was accommodated in the thermostatic chamber 110. If it is determined that the predetermined time has elapsed, in step S21, the transport unit 120 transports the culture vessel 10 to the imaging position. That is, the conveyance unit 120 places the culture container 10 between the imaging unit 30 and the drawing surface 20. More specifically, the transport unit 120 has the culture container 10 in a position and orientation where the culture surface 11 overlaps the drawing surface 20 in a state where the liquid such as the enzyme agent is removed from the culture surface 11 within the imaging field of the imaging unit 30. Positioning.

In step S22, the imaging unit 30 captures an image of the pattern 21 drawn on the drawing surface 20 through the top plate 10A and the bottom plate 10B of the culture vessel 10, and generates image data of the pattern 21. The imaging unit 30 supplies the generated image data of the pattern 21 to the determination unit 40.

Here, FIG. 8A and FIG. 8B are diagrams each illustrating an example of the image of the pattern 21 acquired by the imaging unit 30 in step S22. 8A and 8B show images IMG1 and IMG2 of the pattern 21 that have passed through the culture surface 11, respectively.

When imaging the pattern 21 in the imaging unit 30, the transport unit 120 first starts the culture container so that the side BC of the culture surface 11 is positioned vertically below the side AD as shown in FIG. 8A. 10 positioning is performed. In the present embodiment, imaging of the pattern 21 by the imaging unit 30 is performed in a state where the enzyme agent 210 is accommodated in the culture vessel 10. In this case, the pattern 21 in the vicinity of the side BC is blocked by the enzyme agent 210 moved to the vicinity of the side BC of the culture surface 11. Since the determination part 40 determines the presence or absence of the cell adhering to the culture surface 11 based on the image of the pattern 21, when the pattern 21 in the vicinity of the side BC is blocked by the enzyme agent 210, the determination unit 40 is in the vicinity of the side BC. There is a possibility that it is difficult to appropriately determine the presence or absence of cells by the determination unit 40.

Therefore, after the imaging unit 30 acquires the image IMG1 illustrated in FIG. 8A, the transport unit 120 performs culture so that the side AD of the culture surface 11 is positioned vertically below the side BC as illustrated in FIG. 8B. The container 10 is turned upside down. Thereby, the enzyme agent 210 moves to the vicinity of the side AD of the culture surface 11. In this state, the imaging unit 30 captures the pattern 21 again.

The determination unit 40 determines the presence or absence of cells attached to the culture surface 11 using both the image IMG1 shown in FIG. 8A and the image IMG2 shown in FIG. 8B. Thereby, the determination unit 40 can appropriately determine the presence / absence of cells attached to the culture surface 11 over the entire culture surface 11. The determination unit 40 synthesizes the image IMG1 illustrated in FIG. 8A and the image IMG2 illustrated in FIG. 8B to reconstruct an image in which the enzyme agent 210 is not depicted, and based on the reconstructed image, the culture is performed. The presence or absence of cells adhering to the surface 11 may be determined. Note that when the imaging unit 30 captures the pattern 21, if the enzyme agent 210 does not block the pattern 21, or even if the enzyme agent 210 blocks the pattern 21, the determination process in the determination unit 40 is not hindered. In some cases, imaging in a state where the orientation of the culture vessel 10 is reversed is unnecessary.

In step S 23, the determination unit 40 determines the presence / absence of cells attached to the culture surface 11 based on the image of the pattern acquired by the imaging unit 30. That is, the determination unit 40 performs each process of the flowchart shown in FIG.

When the determination unit 40 determines that “there are cells attached to the culture surface 11”, that is, when it is determined that cells remain on the culture surface 11, the process returns to step S19. That is, in this case, the transport unit 120 accommodates the culture vessel 10 in the thermostatic chamber 110, advances cell detachment, and after a predetermined time has elapsed, captures the pattern 21 by the imaging unit 30, and determination by the determination unit 40 The process is performed again. When it is determined by the determination unit 40 that “no cells are attached to the culture surface 11”, that is, when it is determined that no cells remain on the culture surface 11, the process proceeds to step S 24.

In step S24, the transport unit 120 transports the culture vessel 10 to the cap opening / closing unit 130. In step S 25, the cap opening / closing part 130 opens the cap 12 of the culture vessel 10. In step S 26, the transport unit 120 transports the culture container 10 to the cell collection unit 160.

In step S27, as shown in FIG. 9A, the cell recovery unit 160 inserts a suction nozzle 161 into the inside of the culture container 10 from the circulation port 13 of the culture container 10 exposed by opening the cap 12, and from the culture surface 11 The detached cells are sucked from the suction nozzle 161 and collected in a collection bottle 162 (see FIG. 6). When collecting the cells, the culture vessel 10 may be tilted as shown in FIG. 9A. At this time, it is preferable that the tip of the suction nozzle 161 reaches the lowermost part of the culture vessel 10 in order to suppress cell recovery loss. As shown in FIG. 9B, when the suction nozzle 161 has a tapered shape, a tapered guide hole 14 is provided in the flow port 13 in accordance with the tapered shape of the suction nozzle 161 so that the tip of the suction nozzle 161 is cultured. It becomes possible to position at a specific position inside the container 10.

In step S28, the transport unit 120 transports the culture vessel 10 to the cap opening / closing unit 130. In step S29, the cap opening / closing part 130 closes the cap 12 of the culture vessel 10. In step S 30, the transport unit 120 stores the culture container 10 in the thermostatic chamber 110.

As described above, in the determination apparatus 1 and the cell detachment collection apparatus 2 according to the embodiment of the disclosed technique, the imaging unit 30 places the culture surface 11 of the culture container 10 between the drawing surface 20 and the imaging unit 30. In the arranged state, an image of the pattern 21 drawn on the drawing surface 20 is acquired. When cells are attached to the culture surface 11, in the image of the pattern 21 captured through the culture surface 11, the outline of the pattern 21 (the boundary between the bright part 21 A and the dark part 21 B) Distortion or blur occurs due to the influence of the difference in refractive index from the medium and the scattering of light in the cell. The determination unit 40 determines the presence or absence of cells attached to the culture surface 11 based on the image of the pattern 21 acquired by the imaging unit 30. Specifically, the determination unit 40 is a cell attached to the culture surface 11 based on at least one of distortion and blur in the image portion corresponding to the contour line of the pattern 21 (that is, the boundary between the bright part 21A and the dark part 21B). The presence or absence of is determined. Therefore, even if the cells are transparent or translucent to visible light, it is possible to accurately determine the presence or absence of cells attached to the culture surface 11.

For example, in order to determine the presence / absence of cells adhering to the culture surface 11, if it is intended to observe a plurality of locations on the culture surface 11 in detail, it is assumed that an enormous amount of time is required to obtain the determination result. According to the determination apparatus 1 and the cell detachment collection apparatus 2 according to the embodiment of the disclosed technology, since the image of the pattern 21 drawn on the drawing surface 20 is an analysis target in the determination unit 40, a plurality of culture surfaces 11 are included. Compared with the case where the location is observed in detail, the burden of the determination process in the determination unit 40 can be reduced, and the determination result can be obtained in a short time.

In addition, by including the entire culture surface 11 of the culture vessel 10 within the imaging field of the imaging unit 30, when determining the presence or absence of cell attachment on the entire culture surface 11, the number of imaging by the imaging unit 30 is reduced. be able to. That is, it becomes possible to determine the presence or absence of cells for the entire culture surface 11 from only one image acquired by the imaging unit 30 or only two images in which the orientation of the culture container 10 is in an inverted relationship. .

Further, since the pattern 21 includes a repeated pattern in which the bright portions 21A and the dark portions 21B are alternately arranged, the determination process in the determination unit 40 can be simplified, and the processing time can be shortened. .

Further, when the pattern 21 is imaged by sandwiching the culture container 10 between the imaging unit 30 and the drawing surface 20 by making the area of the pattern 21 larger than the area of the culture surface 11 of the culture container 10, Strict alignment of the culture vessel 10 with respect to the pattern 21 is not necessary.

Further, the imaging of the pattern 21 by the imaging unit 30 is performed in a state where the enzyme agent is accommodated in the culture vessel 10. According to this aspect, the processing time can be shortened compared to the case where imaging is performed after the enzyme agent is removed. In addition, when the determination unit 40 determines that “there are cells attached to the culture surface 11”, the cell detachment process can be continued or resumed without adding the enzyme agent again. .

[Modification]
Various modifications can be made to the configuration of the determination apparatus 1 and the cell detachment collection apparatus 2 described above.

FIG. 10A is a diagram showing an example of another method for positioning the culture vessel 10 at the imaging position. When imaging the pattern 21 in the imaging unit 30, as shown in FIG. 10A, the culture vessel 10 may be positioned using an installation table 170.

The installation table 170 has a guide 171 for installing the culture vessel 10 at a predetermined position on the installation table 170. Further, the surface of the installation table 170 that contacts the culture surface 11 is the drawing surface 20. By positioning the culture vessel 10 on the installation table 170 along the guide 171, the alignment between the culture vessel 10 and the drawing surface 20 (the pattern 21) is completed. Further, in a state where the culture vessel 10 is installed on the installation table 170 along the guide 171, the culture surface 11 is parallel to the vertical direction and the pattern 21 is blocked by the enzyme agent 210 accommodated in the culture vessel 10. The area is minimized. The installation table 170 may be configured to be rotatable around a rotation shaft 172 as shown in FIG. 10B.

FIG. 11 is a diagram showing another example of the configuration of the culture vessel 10. The culture vessel 10 has a storage space 15 for the enzyme agent 210 at its end. When the culture container 10 is positioned at the imaging position, the enzyme agent 210 accommodated in the culture container 10 flows into the accommodation space 15. Thereby, when imaging the pattern 21 by the imaging unit 30, the area of the region where the enzyme agent 210 covers the culture surface 11 (that is, the area of the region where the pattern 21 is blocked by the enzyme agent 210) can be reduced. .

12A, 12B, and 12C are diagrams illustrating an example of another method for removing the enzyme agent 210 from the culture surface 11 when the image 21 is imaged by the imaging unit 30. FIG. In the example shown in FIGS. 12A to 12C, the culture vessel 10 includes a frame 16, a plate member 17, a support member 18, and a partition member 19. A region inside the partition member 19 on one surface of the plate member 17 is a culture surface 11, and a drawing surface 20 on which a pattern 21 is drawn is provided on the surface of the plate member 17 opposite to the culture surface 11. It has been. The partition member 19 is a member that defines the range of the culture surface 11. That is, the range in which the cells 200 spread by proliferation is limited to the area inside the partition member 19. The plate-like member 17 can move in the vertical direction as the push-up pin 180 moves up and down. The partition member 19 is disposed inside the push-up pin 180. That is, the culture surface 11 is disposed inside the push-up pin 180.

FIG. 12A shows a state in which a peeling process for peeling the cells 200 adhered to the culture surface 11 using the enzyme agent 210 is performed. In this state, the push-up pin 180 is in the lowered position, and the plate-like member 17 is supported by the support member 18. The enzyme agent 210 covers the culture surface 11.

When imaging the pattern 21 drawn on the drawing surface 20 in the imaging unit 30, the push-up pin 180 rises and pushes the plate-like member 17 as shown in FIG. 12B. As a result, the plate member 17 is separated from the support member 18, and a gap is formed between the support member 18 and the plate member 17. The enzyme agent 210 that has covered the culture surface 11 passes through the gap formed between the support member 18 and the plate-like member 17 and moves to the bottom surface side of the frame body 16. Thereby, the enzyme agent 210 is removed from the culture surface 11.

Thereafter, as shown in FIG. 12C, the push-up pin 180 is lowered, and the plate member 17 is supported by the support member 18. In this state, the image 21 is imaged by the imaging unit 30. The imaging unit 30 is disposed above the plate-like member 17.

In this way, by removing the enzyme agent 210 from the culture surface 11 by the lifting and lowering operation of the plate-like member 17, in the imaging of the pattern 21 by the imaging unit 30, imaging in a state where the direction of the culture vessel 10 is reversed can be performed. It becomes unnecessary. Further, by arranging the culture surface 11 inside the push-up pin 180, the push-up pin 180 can be excluded from the imaging field when the image of the pattern 21 is picked up.

On the other hand, the removal of the enzyme agent 210 from the culture surface 11 may be realized by image processing. Specifically, the pattern 21 is imaged after cell detachment using an enzyme agent, and then the culture vessel 10 is vibrated to further image the pattern. A cell floating in the enzyme agent is identified from the difference between the two images acquired by these two imaging operations, and the identified cell is removed from one of the two images. Thereby, the image similar to the image acquired in the state which removed the enzyme agent 210 from the culture surface 11 is acquirable.

FIG. 13 is a diagram showing an example of another method for recovering cells detached from the culture surface 11 of the culture vessel 10 by the separation treatment. As shown in FIG. 13, the cells detached by the separation treatment may be discharged from the distribution port 13 by inclining the culture container 10 and directly poured into the recovery bottle 162. In this case, in order to suppress dripping from the circulation port 13, the shape of the circulation port 13 may be processed into a shape in which dripping does not easily occur.

FIG. 14 is a diagram illustrating another example of the configuration of the determination apparatus 1. The drawing surface 20 is constituted by, for example, the surface of a light-transmitting member, a portion where the light transmittance of the drawing surface 20 is reduced is a dark portion 21B, and a portion where the light transmittance of the drawing surface 20 is maintained is a bright portion. In the case of 21A, it is preferable to arrange the light source 50 on the opposite side of the imaging unit 30 with the drawing surface 20 in between, and irradiate the drawing surface 20 with light emitted from the light source 50. Thereby, in the image of the pattern 21 acquired by the imaging part 30, the contrast of the bright part 21A and the dark part 21B can be enlarged, and the precision of the determination process in the determination part 40 can be improved. The light source 50 is preferably a surface light source such as an organic EL (Electro-Luminescence) illumination capable of irradiating light with substantially uniform luminance over the entire drawing surface 20. When a point light source such as a halogen lamp is used as the light source 50, it is preferable to arrange a diffusion plate 51 that diffuses light from the light source 50 between the light source 50 and the drawing surface 20, as shown in FIG.

Further, the pattern 21 drawn on the drawing surface 20 may be formed by light emission by a light emitting element. In this case, the bright portion 21A may be formed by light emission with relatively high luminance, and the dark portion 21B may be formed by light emission (or non-light emission) with relatively low luminance.

FIG. 15 is a diagram illustrating another example of the configuration of the determination apparatus 1. As shown in FIG. 15, the drawing surface 20 may be configured by a display surface of the display panel 60. That is, the drawing surface 20 includes a plurality of pixels arranged in a matrix. When the display panel 60 is a transmissive type such as a liquid crystal panel, for example, the pattern 21 is formed by controlling the light transmission state in each pixel. On the other hand, when the display panel 60 is a self-luminous type such as an organic EL panel, for example, the pattern 21 is formed by controlling the light emission state of each pixel. As described above, the drawing surface 20 is constituted by the display surface of the display panel 60, whereby the pattern of the pattern 21 drawn on the drawing surface 20 can be appropriately changed.

FIG. 16 is a diagram illustrating another example of the configuration of the determination apparatus 1. The determination device 1 may include a derivation unit 70. The deriving unit 70 derives the area of the region where the cells are attached to the culture surface 11 based on the image data of the pattern 21 generated by the imaging unit 30. The derivation unit 70 may be configured to include the same computer as the computer 500 (see FIG. 5) that configures the determination unit 40.

FIG. 17A is a flowchart illustrating an example of a flow of derivation processing performed in the derivation unit 70 to derive the area of a region where cells are attached to the culture surface 11.

In step S41, the derivation unit 70 acquires the image data of the pattern 21 generated by the imaging unit 30.

In step S42, the derivation unit 70 analyzes the image data of the pattern 21 and specifies a region where the outline of the pattern 21 is distorted or blurred.

In step S43, the derivation unit 70 derives the area of the region specified in step S42 as the area of the region where the cells are attached to the culture surface 11. When the plurality of regions are specified in step S42, the deriving unit 70 derives the value obtained by adding the areas of the plurality of regions as the area of the region where the cells are attached to the culture surface 11.

When the determination unit 40 determines that the area derived by the deriving unit 70 is larger than a predetermined threshold, it determines that “there are cells attached to the culture surface 11”. On the other hand, if the determination unit 40 determines that the area derived by the deriving unit 70 is smaller than the predetermined threshold, it determines that “there is no cell attached to the culture surface 11”.

FIG. 17B is a flowchart illustrating another example of the flow of the derivation process performed in the derivation unit 70. The flowchart shown in FIG. 17B is different from the flowchart shown in FIG. 17A in that the process of step S44 is added.

In step S44, the deriving unit 70 derives the ratio of the area derived in step S43 to the area of the culture surface 11 (that is, the area ratio of the region where the cells are attached to the culture surface 11).

When the determination unit 40 determines that the ratio (area ratio) derived by the deriving unit 70 is greater than a predetermined threshold, it determines that “there are cells attached to the culture surface 11”. On the other hand, if the determination unit 40 determines that the ratio (area ratio) derived by the deriving unit 70 is smaller than a predetermined threshold, it determines that “there are no cells attached to the culture surface 11”.

Thus, by determining the presence or absence of cells adhering to the culture surface 11 based on the area of the region where the cells are adhering to the culture surface 11 or its ratio (area ratio), the cell recovery efficiency and Therefore, it is possible to perform more accurate determination in consideration of the processing cost.

FIG. 18 is a diagram illustrating another example of a determination method using the determination device 1. As illustrated in FIG. 18, a plurality of culture vessels 10 may be disposed in a stacked state between the drawing surface 20 and the imaging unit 30. According to this aspect, it is possible to determine whether or not cells are attached to any of the culture surfaces of the plurality of culture containers 10.

The disclosure of Japanese Patent Application No. 2018-027904 filed on February 20, 2018 is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards described in this specification are as much as if each document, patent application, and technical standard were specifically and individually described to be incorporated by reference. , Incorporated herein by reference.

Claims

A drawing surface on which a pattern is drawn, and
An imaging unit having an imaging field of view set on the drawing surface;
Adhering to the culture surface based on the image of the pattern acquired by the imaging unit in a state where a culture surface having light permeability of a culture vessel for culturing cells is disposed between the drawing surface and the imaging unit A determination unit for determining the presence or absence of cells that are
A determination device including:
The determination apparatus according to claim 1, wherein the determination unit determines the presence or absence of cells attached to the culture surface based on at least one of distortion and blur in an image portion corresponding to the outline of the pattern.
The determination apparatus according to claim 1, wherein the pattern includes a bright part and a dark part arranged adjacent to each other.
The determination apparatus according to claim 3, wherein the pattern has a repetitive pattern in which the bright portions and the dark portions are alternately arranged.
The determination apparatus according to claim 1, further comprising a deriving unit that derives an area of a region where cells are attached to the culture surface.
The determination apparatus according to any one of claims 1 to 4, further comprising a deriving unit that derives a ratio of an area of a region where the cell is attached to the culture surface to an area of the culture surface.
The part of the said pattern has light transmittance, The light source arrange | positioned on the opposite side of the said imaging part on both sides of the said drawing surface is further included in any one of Claims 1-6. Judgment device.
The determination device according to claim 1, wherein the pattern is formed by a light emitting element.
The drawing surface includes a plurality of pixels arranged in a matrix,
The determination apparatus according to any one of claims 1 to 8, wherein the pattern is formed by controlling a light transmission state or a light emission state of each of the plurality of pixels.
The determination device according to any one of claims 1 to 9, wherein the imaging unit acquires the image of the pattern in a state where the entire culture surface is included in an imaging field of view.
A determination apparatus according to any one of claims 1 to 10,
A thermostat having a storage space for storing the culture vessel, and maintaining a constant temperature of the storage space;
A transport unit for transporting the culture vessel;
A chemical solution adding unit for adding a chemical solution for separating cells adhering to the culture surface to the culture vessel; a chemical solution removing unit for removing the chemical solution from the culture vessel;
A cell recovery unit for recovering cells detached from the culture surface;
A cell detachment and recovery apparatus.
Set the imaging field of view of the imaging unit on the drawing surface on which the pattern is drawn,
In a state where a culture surface of a culture vessel for culturing cells is disposed between the drawing surface and the imaging unit, an image of the pattern is acquired by the imaging unit,
A determination method for determining the presence or absence of cells attached to the culture surface based on the image of the pattern.